ES2525492T3 - Procedure for preparing an enriched IgG composition from plasma - Google Patents

Abstract

A process for preparing an enriched IgG composition from plasma, the process comprising the steps of: (a) precipitating a plasma fraction devoid of cryoprecipitate, in a first precipitation stage, with alcohol between about 6% and about a 10% at a pH between about 7.0 and about 7.5 to obtain a first precipitate and a first supernatant; (b) precipitate the IgG of the first supernatant, in a second precipitation stage, with alcohol between about 20% and about 25% at a pH between about 6.7 and about 7.3 to form a second precipitate; (c) resuspend the second precipitate to form a suspension; (d) precipitate the IgG of the suspension formed in step (c), in a third stage of precipitation, with alcohol between about 22% and about 28% at a pH between about 6.7 and about 7.3 to form a third precipitate; (e) resuspend the third precipitate to form a suspension; and (f) separating the soluble fraction from the suspension formed in step (e), thereby forming an enriched IgG composition, in which at least one of the first precipitation stage, second precipitation stage, or third stage of precipitation comprises the addition by spraying of alcohol.

Description

DESCRIPTION

Procedure for preparing an IgG composition enriched from plasma

Background of the invention 5

Human plasma immunoglobulin products were first used in 1952 to treat an immunodeficiency. Initially, intramuscular or subcutaneous administration of the immunoglobulin G isotype (IgG) were the procedures of choice. However, to inject greater amounts of IgG necessary for effective treatment of various diseases, intravenously administrable products were developed with a lower concentration of IgG (50 mg / ml). Normally, intravenous immunoglobulin (IVIG) contains the combined immunoglobulin G (IgG) immunoglobulins from the plasma of more than 1,000 blood donors. Typically, they contain more than 95% unmodified IgG, which has intact Fc-dependent effector functions, and only minimal amounts of immunoglobulin A (IgA) or immunoglobulin M (IgM), IVIGs are purified, sterile IgG products, used primarily in the treatment of three main categories of medical conditions: (1) immunodeficiencies such as X-linked agammaglobulinemia, hypogammaglobulinemia (primary immunodeficiencies), and acquired immunity deficiencies (secondary immunodeficiencies), with low antibody levels; (2) inflammatory and autoimmune diseases; and (3) acute infections.

Specifically, many people with primary immunodeficiency disorders lack the necessary antibodies to resist infection. In certain cases, these deficiencies can be supplemented by the infusion of purified IgG, commonly through intravenous administration (i.e., IVIG treatment). Several primary immunodeficiency disorders are commonly treated in this way, including X-linked agammaglobulinemia (XLA), common variable immunodeficiency (CVID), hyper-IgM syndrome (HIM), severe combined immunodeficiency (SCID), and some subclass deficiencies from IgG (Blaese and Winkelstein, J. Patient & Family 25 Handbook for Primary Immunodeficiency Diseases. Towson, MD: Immune Deficiency Foundation; 2007).

Although IVIG treatment can be very effective in treating primary immunodeficiency disorders, this treatment is only a temporary replacement for antibodies that are not produced in the body, rather than a cure for the disease. Consequently, patients dependent on IVIG treatment require repeated doses, typically 30, approximately once a month for life. This need represents a great demand for continued production of IVIG compositions. However, unlike other biological products that are produced through in vitro expression of recombinant DNA vectors, IVIG is fractionated from human blood and plasma donors. Therefore, IVIG products cannot be increased by simply increasing production volume. Rather, the level of commercially available IVIG is limited by the available supply of blood and plasma donations.

Several factors drive the demand for IVIG, including the acceptance of IVIG treatments, the identification of additional indications for which IVIG treatment is effective, and the increase in patient diagnosis and the prescription of IVIG. Notably, IVIG's global demand has more than quadrupled since 1990 and continues to increase 40 today at an annual rate of between approximately 7% and 10% (Robert P., Pharmaceutical Policy and Law, 11 (2009) 359 -367). For example, the Australian National Blood Authority reported that IVIG demand in Australia grew by 10.6% during the 2008-2009 budget year (National Blood Authority Australia Annual Report 2008-2009).

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Due in part to the increase in global demand and fluctuations in the available supply of immunoglobulin products, several countries, including Australia and England, have implemented demand management programs to protect supplies of these products for patients with increased demand during times of product shortages.

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It is reported that in 2007, 26.5 million liters of plasma were fractionated, generating 75.2 metric tons of IVIG, with an average production yield of 2.8 grams per liter (Robert P., supra). This same report estimated that IVIG's global yields were expected to increase by approximately 3.43 grams per liter in 2012. However, due to the continued growth of IVIG's global demand, extrapolated between approximately 7% and 13% annually. Between now and 2015, an additional 55 improvement in total IVIG performance will be needed to meet global demand.

Various IVIG preparation procedures are used by commercial suppliers of IVIG products. A common problem with current IVIG production procedures is the substantial loss of IgG during the purification procedure, which is estimated to be at least 30% to 35% of the total IgG content of the starting material. A challenge is to maintain the quality of viral inactivation and the lack of impurities that can cause adverse reactions, while strengthening the performance of IgG. At current IVIG production levels, what is considered small increases in performance are, in fact, highly significant. For example, at production levels in 2007, a 2% increase in efficiency, equal to an additional 56 milligrams per liter, would generate an additional 1.5 metric tons of IVIG. 65

Documents DE 100 08 619, EP 0 893 450, US 4 550 019 disclose methods for the manufacture of immunoglobulin compositions. The same applies to: documents US 6 150 471, US 4 499 073; Cammerata-P.B., Cohen-P.P. Fractionation and Properties of Glutamic-Oxalacetic Transaminase. jbc.org. 1981. 53-62. Curling-J-M. Methods of Plasma Protein Fractionation. Academic Press 1980.

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In the fourth installment of a series of influential articles published on the preparation and properties of whey and plasma proteins, Cohn et al. (J. Am. Chem. Soc., 1946, 68 (3): 459-475) described for the first time a method for alcohol fractionation of plasma proteins (procedure 6), which allows the isolation of a fraction enriched in IgG from human plasma. Several years later, Oncley et al. (J. Am. Chem. Soc., 1949, 71 (2): 541-550) expanded Cohn's procedures by publishing a procedure 10 (procedure 9) that resulted in the isolation of a purer IgG preparation.

These procedures, although they lay the foundation for the entire plasma-derived blood factor industry, were unable to provide IgG preparations that had sufficiently high concentrations for the treatment of several immunorelated diseases, including Kawasaki syndrome, immune thrombocytopenic purpura and primary immunodeficiencies. . As such, additional methodologies were developed that employed various techniques, such as ion exchange chromatography, to provide IgG formulations with higher purity and higher concentration. Hoppe et al. (Munch Med Wochenschr 1967 (34): 1749-1752) and Falksveden (Swedish Patent No. 348942) and Falksveden and Lundblad (Methods of Plasma Protein Fractionation 1980) were among the first to use ion exchange chromatography for this purpose. twenty

Several modern procedures employ a precipitation stage, such as precipitation with caprylate (Lebing et al., Vox Sang 2003 (84): 193-201) and ethanol precipitation of Cohn fraction (I +) II + III (Tanaka et al. ., Braz J Med Biol Res 2000 (33) 37-30) coupled to column chromatography. More recently, Teschner et al. (Vox Sang, 2007 (92): 42-55) have described a procedure for the production of a 10% IVIG product in which, firstly, the cryoprecipitate is removed from the combined plasmas and then A fractionation with cold modified Cohn-Oncley ethanol is performed, followed by the intermediate S / D treatment, ion exchange chromatography, nanofiltration, and optionally ultrafiltration / diafiltration.

However, despite the improvement in purity, safety and performance provided by these IgG manufacturing processes, a significant amount of IgG is still lost during the manufacturing process. For example, Teschner et al. They reported that their procedure resulted in an increase in IgG yield of 65% (Teschner et al., supra). As reported during several meetings on plasma products, the average yield for large-scale IgG preparation, such as Baxter, CSL Behring, Upfront Technology, Cangene, Prometric BioTherapeutics, and the Finnish Red Cross, varies from about 61% to 35 about 65% in the final container. This represents a loss of at least about one third of the IgG present in the combined plasma fraction during the manufacturing process.

As such, there is a need for improved and more efficient procedures for manufacturing IVIG products. The present invention satisfies these and other needs by providing IVIG manufacturing processes that produce yields that are at least 6 to 10% higher than currently attainable, as well as IVIG compositions provided therefrom.

Brief summary of the invention

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In one aspect, the present invention provides methods for preparing enriched IgG compositions (eg, IVIG compositions) from plasma according to claim 1. Advantageously, the methods provided herein provide significant improvements over Manufacturing processes of the state of the art to prepare IVIG compositions. For example, the procedures provided herein allow an increase in IgG yields in the final bulk composition without losing the purity required for intravenous administration.

In one aspect, a method is provided for preparing a plasma-enriched IgG composition comprising the steps of (a) precipitating a plasma fraction devoid of cryoprecipitate, in a first precipitation stage, with alcohol between about 6% and about 10% at a pH of between about 7.0 and about 7.5 to obtain a first precipitate and a first supernatant, (b) precipitate the IgG of the first supernatant, in a second precipitation stage, with alcohol between about 20% and about 25% at a pH between about 6.7 and about 7.3 to form a second precipitate, (c) resuspend the second precipitate to form a suspension, (d) precipitate the IgG from the suspension formed in step (c), in a third precipitation stage, with alcohol between about 60-22% and about 28% at a pH between about 6.7 and about 7.3 pa ra forming a third precipitate, (e) resuspending the third precipitate to form a suspension, and (f) separating the soluble fraction from the suspension formed in step (e), thereby forming an enriched IgG composition, in which at least one of the first precipitation stage, second precipitation stage, or third precipitation stage comprises the alcohol addition by spraying. In one embodiment, alcohol is added in the first stage 65 of spray precipitation. In another embodiment, alcohol is added in the second precipitation stage

by spraying In yet another embodiment, alcohol is added in the third stage of spray precipitation.

In certain embodiments, the pH of one or more solutions can be adjusted by the addition of a pH modifying agent by spraying. In related embodiments, the pH of at least one of the first 5 precipitation stage, second precipitation stage or third precipitation stage is achieved by the addition of a pH modifying solution after the alcohol addition, or before and after the addition of alcohol, during and after the addition of alcohol, or before, during, and after the addition of alcohol. In yet another related embodiment, the pH of a precipitation step can be maintained throughout the precipitation reaction by continuously adjusting the pH. 10

In a specific embodiment, the pH of the first precipitation stage is adjusted after the addition of alcohol by spray addition of a pH modifying agent. In another specific embodiment, the pH of the second precipitation stage is adjusted after the addition of alcohol by spray addition of a pH modifying agent. In yet another specific embodiment, the pH of the third stage of precipitation is adjusted after the addition of alcohol by spray addition of a pH modifying agent.

Additionally, the preparatory procedures provided herein may further comprise an ion exchange chromatography step (ie, anion exchange and / or cation exchange chromatography), a nanofiltration stage, an ultrafiltration / diafiltration stage, or any another technique of purification suitable to further enhance the purity or quality of IVIG preparations.

In another aspect, a method is provided for preparing an IgG composition enriched from plasma, comprising the steps of (a) adjusting the pH of a plasma fraction devoid of cryoprecipitate up to or up to about 7.0, (b) adjusting the ethanol concentration of the plasma fraction devoid of 25 cryoprecipitate from step (a) up to about 25% (v / v) at a temperature between or between about -7 ° C and about -9 ° C, forming this mode a mixture, (c) separating liquid and precipitate from the mixture of step (b), (d) resuspending the precipitate from step (c) with a buffer containing phosphate and acetate, in which the pH of the buffer is adjust with or with approximately 600 ml of glacial acetic acid per 1000 l of buffer, thereby forming a suspension, (e) mixing finely divided silicon dioxide (SiO2) with the suspension of step (d) for at least approximately 30 minutes, (f) filt Rare the suspension with a filter-press, thereby forming a filtrate, (g) wash the filter-press with at least 3 dead volumes of filter-press from a buffer containing phosphate and acetate, in which the pH of the buffer it is adjusted with or with approximately 150 ml of glacial acetic acid per 1000 l of buffer, thereby forming a wash solution, (h) combining the filtrate of step (f) with the washing solution of step (g) , thereby forming a solution, and treating the solution with a detergent, (i) adjust the pH of the solution of step (h) to or up to about 7.0 and add ethanol to a final concentration of one or about 25%, thereby forming a precipitate, (j) separating liquid and precipitate from the mixture of step (i), (k) dissolving the precipitate in an aqueous solution comprising a solvent or detergent and maintaining the solution for at minus 60 minutes, (1) run the solution after the stage (k ) through a cation exchange chromatography column and eluting the absorbed proteins in the column in an eluate, (m) passing the eluate from step (1) through an anion exchange chromatography column to generate a effluent, (n) passing the effluent from stage (m) through a nanofilter to generate a nanofiltrate, (or) passing the nanofiltrate from stage (n) through an ultrafiltration membrane to generate an ultrafiltrate, and (p) diafiltering the ultrafiltrate of step (o) against a diafiltration buffer to generate a diafiltrate having a protein concentration between about 8% (w / v) and about 12% (w / v ), thereby obtaining a concentrated IgG composition.

In another aspect, the present invention provides aqueous IgG compositions prepared by the methods described herein. In general, IgG compositions have a high purity (for example, 50 IgG content at least 95%, 98%, 99%, or greater), contain protein concentrations of between about 20 g / l and about 200 g / l, and contain extremely low levels of common IVIG contaminants, such as IgG, IgM, fibrinogen, transferrin, ACA, amidolytic activity, PKA, and the like.

In still another aspect, pharmaceutical IgG compositions and formulations suitable for use in IVIG treatments are provided. Pharmaceutical formulations have a high purity (for example, IgG content of at least 98%, 99%, or greater), contain protein concentrations of between about 20 g / l and about 200 g / l, and contain extremely low levels of common IVIG contaminants, such as IgG, IgM, fibrinogen, transferrin, ACA, amidolytic activity, PKA, and the like. In general, pharmaceutical compositions are formulated appropriately for intravenous administration (ie, for treatment with IVIG), subcutaneous administration or intramuscular administration.

In another aspect, the present invention provides methods for treating an immunodeficiency, autoimmune disease or acute infection, in a human being in need thereof, the method comprising administering a pharmaceutical composition comprising herein described. Non-limiting examples of 65 diseases and conditions that can be treated or managed through the procedures provided in the

This document includes bone marrow allograft transplantation, chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura (ITP), childhood HIV, primary immunodeficiencies, Kawasaki disease, chronic inflammatory demyelinating polyneuropathy (CIDP), kidney transplant with a high antibody donor receptor or AB0 incompatible, chronic fatigue syndrome, Clostridium difficile colitis, dermatomyositis and polymyositis, Graves ophthalmopathy, Guillain-Barre syndrome, muscular dystrophy, inclusion body myositis, Lambert-Eaton syndrome, lupus 5 erythematosus, multifocal motor neuropathy, sclerosis multiple (MS), myasthenia gravis, neonatal alloimmune thrombocytopenia, Parvovirus B19 infection, pemphigus, post-transfusion purpura, renal transplant rejection, spontaneous / natural abortion, rigid man syndrome, opsoclonia-myoclonus, severe septicemia or septic shock in patients Critical adults, epidermal necrolysis toxic, chronic lymphocytic leukemia, multiple myeloma, X-linked agammaglobulinemia, hypogammaglobulinemia, primary immunodeficiency, RRMS, 10 Alzheimer's disease, and Parkinson's disease.

Brief description of the drawings

Figure 1: IgG concentration determined by ELISA (▲) and nephelometry (●) and total protein (■) concentration present in the filtration wash of fraction II + III as a function of the number of dead buffer volumes used to wash the filtration device after filtration.

Figure 2: Average PKA activity in dissolved fractions of precipitate G after extraction and rinsing at pH 3.8 to 5.0 by the addition of acetic acid in the presence and absence of treatment with Aerosil (silicon dioxide) .

Figure 3: Average fibrinogen content in dissolved fractions of precipitate G after extraction and rinsing at pH 3.8 to 5.0 by the addition of acetic acid in the presence and absence of treatment with Aerosil (silicon dioxide). 25

Figure 4: Average amidolytic activity in dissolved fractions of precipitate G after extraction and rinsing at pH 3.8 to 5.0 by the addition of acetic acid in the presence and absence of treatment with Aerosil (silicon dioxide).

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Figure 5: Amidolytic activity in dissolved fractions of precipitate G extracted and clarified at pH 3.8 to 7.8 after incubation for two weeks at 4 ° C (♦) or for an additional week at room temperature (■).

Figure 6: PKA activity in dissolved fractions of precipitate G extracted and clarified at pH from 3.8 to 7.8.

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Figure 7: Difference in the purity of the filtrate of fraction II + III modified with and without combustion silica treatment. Cellulose acetate electrophoresis chromatography of the modified fraction II + III filtrate (A) rinsed only with filter aid and (B) rinsed after combustion silica treatment.

Figure 8: Change in the pH of 6.9 of the supernatant of fraction II + III after the addition of alcohol in precipitation in the manufacture of large-scale IgG.

Figure 9: Amount of glacial acetic acid versus pH in the precipitate for the extraction of the precipitate of the modified fraction II + III.

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Figure 10: Amount of glacial acetic acid versus pH in the subsequent wash buffer of the modified fraction II + III suspension filter.

Definitions

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As used herein, an "antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, that specifically binds and recognizes an analyte (antigen). Recognized immunoglobulin genes include the genes from the constant region kappa, lambda, alpha, gamma, delta, epsilon and mu, as well as myriad genes from the variable region of immunoglobulin. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the classes of immunoglobulins, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having a "light" (approximately 25 kD) and a "heavy" (approximately 50-70 kD) chain. The N-terminal end of each chain defines a variable region of approximately 100 to 110 or more 60 amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

As used herein, the term "ultrafiltration (UF)" encompasses a variety of membrane filtration procedures in which hydrostatic pressure forces a liquid against a semipermeable membrane. The 65 suspended solids and solutes of high molecular weight are retained, while water and low solutes

Molecular weight pass through the membrane. This separation procedure is often used to purify and concentrate macromolecular solutions (103-106 Da), especially protein solutions. Several ultrafiltration membranes are available depending on the size of the molecules they retain. Typically, ultrafiltration is characterized by a membrane pore size between 1 and 1000 kDa and operating pressures between 0.01 and 10, and is particularly useful for separating colloids as proteins from small molecules such as sugars and salts. 5

As used herein, the term "diafiltration" is performed with the same membranes as ultrafiltration and is a tangential flow filtration. During diafiltration, the buffer is introduced into the recycling bin while the filtrate is removed from the operation of the unit. In procedures in which the product is in the retentate (eg IgG), the diafiltration washes the components of the combined product 10 in the filtrate by washing, thereby exchanging buffers and reducing the concentration of unwanted species.

As used herein, the term "approximately" indicates an approximate range of plus or minus 10% of a specific value. For example, the language "approximately 20%" encompasses an interval of 18-22%. fifteen

As used herein, the term "mixing" describes an act of causing an equitable distribution of two or more different compounds or substances in a solution or suspension by any form of agitation. The complete equitable distribution of all ingredients in a solution or suspension is not required as a result of "mixing" as the term is used in this application. twenty

As used herein, the term "solvent" encompasses any liquid substance that can dissolve or disperse one or more other substances. A solvent may be inorganic in nature, such as water, or it may be an organic liquid, such as ethanol, acetone, methyl acetate, ethyl acetate, hexane, petroleum ether, etc. As used in the term "solvent detergent treatment", solvent indicates an organic solvent (for example, tri-N-butyl phosphate), which is part of the solvent detergent mixture used to inactivate the lipid-enveloped viruses in solution.

As used herein, the term "detergent" is used interchangeably with the term "surfactant" or "surface acting agent" in this application. Typically, the surfactants are organic compounds that are amphiphilic, that is, they contain both hydrophobic groups ("tails") and hydrophilic groups ("heads"), which make the surfactants soluble in both organic solvents and water. A surfactant can be classified by the presence of formally charged groups in its head. A non-ionic surfactant has no charged groups on its head, while an ionic surfactant carries a net charge on its head. A bipolar surfactant contains a head with two groups with opposite charge. Some examples of common surfactants include: anionic (based on sulfate, sulphonate or carboxylate anions): perfluorooctanoate (PFOA or PFO), perfluorooctane sulfonate (PFOS), sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other sulfate salts alkyl, sodium lauryl ether sulfate (also known as sodium lauryl ether sulfate, or SLES), alkylbenzene sulfonate; cationic (based on quaternary ammonium cations): cetyl-trimethylammonium bromide (CTAB) also called hexadecyltrimethylammonium bromide, and other alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzal chloride BAC), benzethonium chloride (BZT); long chain fatty acids and their salts: including caprylate, caprylic acid, heptanoate, hexanoic acid, heptanoic acid, nanoic acid, decanoic acid, and the like; bipolar (amphoteric): dodecylbetaine; cocamidopropyl betaine; cocoanfoglycinate; Non-ionic: poly (ethylene oxide) alkyl, poly (ethylene oxide) alkyl phenol, copolymers of poly (ethylene oxide) and poly (polypropylene oxide) (commercially known as poloxamers or poloxamines), alkyl polyglucosides, including 45 octylglucoside , decylmaltósido, fatty alcohols (for example, cetyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates (Tween 20, Tween 80, etc.), Triton detergents, and dodecyldimethylamine oxide.

As used herein, the term "intravenous IgG" or "IVIG" treatment refers, in general, to a therapeutic procedure of intravenous, subcutaneous or intramuscular administration of a composition of 50 IgG immunoglobulins to a patient to treat several conditions such as immunodeficiencies, inflammatory diseases and autoimmune diseases. Typically, IgG immunoglobulins are combined and prepared from plasma. Complete antibodies or fragments can be used. IgG immunoglobulins may be formulated in higher concentrations (eg, greater than 10%) for subcutaneous administration, or may be formulated for intramuscular administration. This is particularly common for specialized IgG preparations 55 that are prepared with titers greater than average for specific antigens (for example, Rho D factor, pertussis toxin, tetanus toxin, botulinum toxin, rabies, etc.). To facilitate the analysis, said IgG compositions formulated subcutaneously or intramuscularly are also included in the term "IVIG" in this application.

By the term "therapeutically effective amount or dose" or "sufficient / effective amount or dose", a dose is meant that produces the effects for which it is administered.The exact dose will depend on the purpose of the treatment, and will be determinable by a skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vol. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999) ; and Remington: The Science and Practice of Pharmacy, 20th edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins.) 65

As used in this application, the term "spray" refers to a means of administering a liquid substance in a system, for example, during an alcohol precipitation stage, such as a fractionation I or II + III precipitation stage. of modified Cohn, in the form of droplets or fine vaporization of the liquid substance. Spraying can be achieved by any pressurized device, such as a container (for example, a spray bottle), which has a spray head or a nozzle and is operated manually or automatically to generate a fine vaporization of a liquid . Typically, spraying is performed while the system receiving the liquid substance is continuously stirred or otherwise mixed to ensure a rapid and equitable distribution of the liquid within the system.

Detailed description of the invention: 10

I. Overview

As routinely practiced in modern medicine, sterile preparations of concentrated immunoglobulins (especially IgG) are used to treat medical conditions that fall into three main classes: 15 immune deficiencies, inflammatory and autoimmune diseases, and acute infections. A commonly used IgG product, intravenous immunoglobulin or IVIG, is formulated for intravenous administration, for example, in an IgG concentration of or about 10%. Concentrated immunoglobulins can also be formulated for subcutaneous or intramuscular administration, for example, in an IgG concentration of or about 20%. To facilitate the analysis, said IgG compositions formulated subcutaneously or intramuscularly are also included in the term "IVIG" in this application.

In certain aspects, the present invention provides methods for the manufacture of IVIG that increase the final yield of the product, still provide IVIG compositions of equal or greater quality and in some cases higher concentrations. In one embodiment, the present invention provides 25 modified Cohn fractionation methods that reduce the loss of IgG in one or more precipitation steps.

In another aspect, the present invention provides IgG compositions prepared in accordance with an improvement in the manufacturing processes provided herein. Advantageously, these 30 compositions are less expensive to prepare than the commercial products currently available due to the improvement in performance provided by the procedures provided herein. In addition, these compositions are as pure, or purer, than compositions made using commercial procedures. Importantly, these compositions are suitable for use in IVIG treatment for immunodeficiencies, inflammatory and autoimmune diseases, and acute infections. In one embodiment, the IgG composition has IgG at or about 10% for intravenous administration. In another embodiment, the IgG composition is of or about 20% for subcutaneous or intramuscular administration.

In another aspect, the present invention provides pharmaceutical compositions and formulations of IgG compositions 40 prepared in accordance with the improvement in manufacturing methodologies provided herein. In certain embodiments, these compositions and formulations provide an improvement in properties compared to other IVIG compositions currently on the market. For example, in certain embodiments, the compositions and formulations provided herein are stable for a prolonged period of time. Four. Five

In yet another aspect, the present invention provides a method for treating immunodeficiencies, inflammatory and autoimmune diseases, and acute infections comprising the administration of an IgG composition prepared using the improvement in the procedures provided herein.

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II. IVIG manufacturing procedures

In general, immunoglobulin preparations according to the present invention can be prepared from any suitable starting material, for example, recovered plasma or source plasma. In a typical example, blood or plasma is collected from healthy donors. Normally, blood is collected from the same animal species as the subject to which the immunoglobulin preparation (typically referred to as "homologous" immunoglobulins) will be administered. Immunoglobulins are isolated from the blood by suitable procedures, such as, for example, precipitation (alcohol fractionation or polyethylene glycol fractionation), chromatographic procedures (ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, etc.) ultracentrifugation, and electrophoretic preparation, and the like. (See, for example, Cohn et al., J. Am. Chem. Soc. 60 68: 459-75 (1946); Oncley et al., J. Am. Chem. Soc. 71: 541-50 (1949) ; Barundem et al., Vox Sang. 7: 157-74 (1962); Koblet et al., Vox Sang. 13: 93-102 (1967); U.S. Patent Nos. 5,122,373 and 5,177. 194; of which the disclosures are incorporated herein by reference in their entirety for all purposes).

In many cases, immunoglobulins are prepared from products containing gamma globulin produced by alcohol fractionation and / or ion exchange chromatography and affinity chromatography procedures.

well known to those skilled in the art. For example, purified Cohn fraction II is commonly used as a starting point for the isolation of immunoglobulins. Typically, the starting Cohn fraction II paste has approximately 95 percent IgG and is composed of the four IgG subtypes. The different subtypes are present in fraction II approximately in the same proportion in which they are found in the combined human plasmas from which they are obtained. Fraction II is further purified before formulation 5 in an administrable product. For example, the paste of fraction II can be dissolved in a solution of purified aqueous alcohol and the impurities can be removed by precipitation and filtration. After final filtration, the immunoglobulin suspension can be subjected to dialysis or diafiltration (for example, using ultrafiltration membranes having a nominal molecular weight limit of less than or equal to 100,000 daltons) to remove the alcohol. The solution can be concentrated or diluted to obtain the desired protein concentration and can be further purified by techniques well known to those skilled in the art.

In addition, additional preparative steps can be used to enrich a particular isotype or immunoglobulin supotype. For example, chromatography with protein A, protein G or protein Sepharose can be used to enrich a mixture of immunoglobulins for IgG, or for specific IgG subtypes. In general, see, Harlow and 15 Lane, Using Antibodies, Cold Spring Harbor Laboratory Press (1999); Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988); and the US patent. UU. No. 5,180,810, of which the disclosures are incorporated herein by reference in their entirety for all purposes.

In contrast to the methods described above, in one aspect, the present invention provides methods of preparing concentrated IgG compositions using a starting material devoid of cryoprecipitate. In general, the procedures provided herein utilize both fractionation steps with modified Cohn-Oncley alcohol and ion exchange chromatography to provide higher IgG yields, while maintaining the same or improved quality as found in the preparations. of commercial IVIGs currently available. For example, in certain embodiments, methods are provided that provide a final bulk IgG composition containing almost 75% of the IgG content found in the starting plasma raw material. These procedures represent an increase of at least 10% to 12% in the total IgG yield over the existing state of the art purification procedures. For example, it is estimated that the GAM-MAGARD® LIQUID manufacturing process provides a final yield of between about 60% and 65% of the IgG content found in the starting material. As such, the procedures provided herein provide a significant improvement over existing IgG purification technologies.

In one embodiment, the present invention provides a purified IgG composition containing at least 70% of the IgG content found in the starting plasma raw material. In another embodiment, a purified IgG composition is provided that contains at least 75% of the IgG content found in the starting plasma raw material. In other embodiments, a purified IgG composition provided herein will contain at least about 65% of the IgG content found in the starting plasma raw material, or at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, or more of the IgG content found in the starting plasma raw material. 40

A. Precipitation fractionation procedures of modified alcoholic / ion exchange chromatography

In one aspect, the present invention provides improved methods for manufacturing IgG compositions suitable for use in IVIG treatment. In general, these procedures provide 45 IgG preparations that have higher yields and comparable purity, if not greater, than the current procedures employed for the production of commercial IVIG products.

In a specific aspect, the present invention provides a process for preparing a plasma concentrated IgG composition, for example, 10% IVIG, the method comprising performing at least one stage of alcohol precipitation and at least one chromatography stage of ion exchange In particular, several stages in the improved preprocessing process are different from the above procedures, for example, the use of 25% ethanol at lower temperatures, addition of ethanol by spraying, adjustment of pH by spraying and the use of silica particles divided.

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In a certain embodiment, the process comprises the steps of (a) precipitating a plasma fraction devoid of cryoprecipitate, in a first precipitation stage, with alcohol between about 6% and about 10% at a pH between about 6.7 and about 7.3 to obtain an IgG-enriched supernatant, (b) precipitate IgG from the supernatant with alcohol between about 20% and about 30% at a lower temperature and at a pH between about 6.7 and about 7.3 60 to form a first precipitate, (c) resuspend the first precipitate formed in step (b) to form a suspension, (d) treat the suspension formed in step (c) with a detergent, (e) precipitate IgG of the suspension with alcohol between about 20% and about 30% at a pH between about 6.7 and about 7.3 to form a second precipitate, (f) resuspend the second precipitate formed in step (e) to form a suspension, (g) treat the suspension formed in step (f) with a solvent and / or detergent, and (h) 65 perform at least one fractionation by ion exchange chromatography preparing from this mode one

concentrated IgG composition. In one embodiment, the process further comprises treating the suspension formed in step (c) with finely divided silica (SiO2) and filtering the solution prior to step (d).

In one embodiment, there is provided a process for preparing a concentrated plasma IgG composition, the process comprising the steps of (a) adjusting the pH of a fraction of plasma devoid of cryoprecipitate to about 7.0, (b) adjust the ethanol concentration of the plasma fraction devoid of cryoprecipitate from step (a) to about or about 25% (v / v) at a temperature between about -5 ° C and about -9 ° C, thereby forming a mixture, in which the concentration of ethanol can be adjusted by spraying, (c) separating liquid and precipitate from the mixture of step (b), (d) resuspending the precipitate from step (c) with a buffer containing phosphate and acetate, in which the pH of the buffer is adjusted with between about 400 and about 700 ml of glacial acetic acid per 1000 l of buffer, thereby forming a suspension, (e) mixing silicon dioxide (SiO2) finely divided with the suspension of step (d) for at least about 30 minutes, (f) filtering the suspension with a filter-press, thereby forming a filtrate, (g) washing the filter-press with at least 3 dead volumes filter press of a buffer containing phosphate 15 and acetate, in which the pH of the buffer is adjusted with approximately 150 ml of glacial acetic acid per 1000 l of buffer, thereby forming a wash solution, (h) combine filtering stage (f) with the washing solution of stage (g), thereby forming a solution, and treating the solution with a detergent, (i) adjusting the pH of the solution of stage (h) to about 7.0 and add ethanol to a final concentration of one or about 25%, thereby forming a precipitate, in which the concentration of ethanol and / or the pH can be adjusted by spraying (j) removing liquid and precipitate from the mixture of step (i), (k) dissolve the precipitate in an aqueous solution comprising a solvent or detergent and maintaining the solution for at least 60 minutes, (1) passing the solution after step (k) through a cation exchange chromatography column and eluting the absorbed proteins in the column in an eluate, (m) passing the eluate from step (1) through an anion exchange chromatography column to generate an effluent (i.e., flow through), (n) passing the effluent of step (m) through a nanofilter to generate a nanofiltrate, (or) pass the nanofiltrate of step (n) through an ultrafiltration membrane to generate an ultrafiltrate, and (p) diafiltrate the ultrafiltrate of step (o) against a diafiltration buffer to generate a diafiltrate having a protein concentration between about 8% (w / v) and about 22% (w / v), thereby obtaining an IgG composition concentrated. In one embodiment, the temperature of step (b) is about 30 of -7 ° C. In a specific embodiment, the suspension buffer in step (d) is adjusted with approximately 600 ml of glacial acetic acid.

In certain embodiments, the diafiltrate will have a protein concentration between about 8% and about 12%, for example, about 8%, or about 9%, 10%, 11%, 35 or 12%. In a preferred embodiment, the diafiltrate will have a protein concentration of one or about 10%. In another preferred embodiment, the diafiltrate will have a protein concentration of one or about 11%. In yet another preferred embodiment, the diafiltrate will have a protein concentration of one or about 12%. In other embodiments, the diafiltrate will have a protein concentration between about 13% and about 17%, for example, about 13%, or about 14%, 15%, 16%, or 17%. In still other embodiments, the diafiltrate will have a protein concentration between about 18% and about 22%, for example, about 18%, or about 19%, 20%, 21%, or 22%. In a preferred embodiment, the diafiltrate will have a protein concentration of one or about 20%. In another preferred embodiment, the diafiltrate will have a protein concentration of one or about 21%. In yet another preferred embodiment, the diafiltrate will have a protein concentration of one or about 22%.

In certain embodiments of the present invention, the methods provided herein may comprise improvements in two or more of the steps of the fractionation process described above. For example, embodiments may include improvements in the precipitation stage, the precipitation stage of the modified fraction II + III, the dissolution stage of the modified fraction II + III, and / or the suspension filtration stage of the modified fraction II + III.

In one embodiment, the improvement made in the first precipitation stage is the addition of alcohol by spraying. In another embodiment, the improvement made in the first precipitation stage is the addition of a pH modifying agent by spraying. In yet another embodiment, the improvement made in the first precipitation stage is the adjustment of the pH of the solution after the addition of the alcohol. In a related embodiment, the improvement made in the first precipitation stage is the maintenance of the pH of the solution during the alcohol addition. In another related embodiment, the improvement made in the first precipitation stage is the maintenance of the pH during the precipitation incubation time by continuously adjusting the pH of the solution. In certain embodiments, the first precipitation stage can be improved by implementing more than one of these improvements. Other improvements that may occur at this stage will be apparent from the section provided below that analyzes the first stage of precipitation (modified fractionation I). By implementing one or more of the improvements described above, a reduced amount of IgG is lost in the precipitate fraction of the first precipitation stage and / or a reduced fraction of 65 IgG is irreversibly denatured during the precipitation stage.

In one embodiment, the improvement made in the precipitation stage of the modified fraction II + III is the addition of alcohol by spraying. In another embodiment, the improvement made in the precipitation stage of the modified fraction II + III is the addition of a pH modifying agent by spraying. In yet another embodiment, the improvement made in the precipitation stage of the modified fraction II + III is the adjustment of the pH of the solution after the alcohol addition. In a related embodiment, the improvement made in the step of precipitation of the modified fraction II + III is the maintenance of the pH of the solution during the alcohol addition. In another related embodiment, the improvement made in the precipitation stage of the modified fraction II + III is the maintenance of the pH during the precipitation incubation time by continuously adjusting the pH of the solution. In another aspect, the precipitation stage of the modified fraction II + III is improved by increasing the alcohol concentration to about or about 25%. In yet another embodiment, the precipitation step of the modified fraction II + III is improved by lowering the incubation temperature to between about -7 ° C and -9 ° C. In certain embodiments, the precipitation stage of the modified fraction II + III can be improved by implementing more than one of these improvements. Other improvements that may occur at this stage will be apparent from the section provided below that analyzes the second stage of precipitation (modified Fractionation II + III). By implementing one or more of the improvements described above, a reduced amount of IgG is lost in the supernatant fraction of the precipitation stage of the modified fraction II + III and / or a reduced fraction of IgG is irreversibly denatured during the precipitation stage

In one embodiment, the improvement made in the dissolution stage of the modified fraction II + III is achieved by increasing the glacial acetic acid content of the dissolution buffer to approximately 0.06%. In another embodiment, the improvement made in the dissolution stage of the modified fraction II + III is achieved by maintaining the pH of the solution during the incubation time of the solution by continuously adjusting the pH of the solution. In another embodiment, the improvement made in the stage of dissolution of the modified faction II + III is achieved by mixing finely divided silicon dioxide (SiO2) with the suspension of fraction II + III before filtration. In certain embodiments, the stage of dissolution of the modified fraction II + III can be improved by implementing more than one of these improvements. Other improvements that may occur at this stage will be apparent from the section provided below that analyzes the stage of dissolution of the modified fraction II + III (Extraction of the precipitate from the modified fraction II + III). By implementing one or more of the improvements described above, an increased amount of IgG is recovered in the suspension of fraction II + III and / or the amount of impurities in the suspension of fraction II + III is reduced. 30

An example improvement made in the filtration step of the suspension of the modified fraction II + III is produced by subsequently washing the filter with at least about 3.6 dead volumes of a solution buffer containing or containing about 150 ml of acid. glacial acetic per 1000 l. Other improvements that may occur at this stage will be apparent from the section provided below that analyzes the suspension filtration stage of the modified fraction II + III (Pretreatment and filtration of the suspension of the modified fraction II + III) . By implementing one or more of the improvements described above, a reduced amount of IgG is lost during the suspension filtration step of the modified fraction II + III.

In one embodiment, the process may comprise an improvement in the first precipitation stage and the precipitation stage of the modified fraction II + III.

In another embodiment, the process may comprise an improvement in the first precipitation stage and the dissolution stage of the modified fraction II + III.

 Four. Five

In another embodiment, the process may comprise an improvement in the first precipitation stage and the suspension filtration stage of the modified fraction II + III.

In another embodiment, the process may comprise an improvement in the precipitation stage of the modified fraction II + III and the dissolution stage of the modified fraction II + III. fifty

In another embodiment, the process may comprise an improvement in the precipitation stage of the modified fraction II + III and the suspension filtration step of the modified fraction II + III.

In another embodiment, the process may comprise an improvement in the dissolution stage of the modified fraction 55 II + III and the suspension filtration step of the modified fraction II + III.

In another embodiment, the process may comprise an improvement in the first precipitation stage, the precipitation stage of the modified fraction II + III and the dissolution stage of the modified fraction II + III.

 60

In another embodiment, the process may comprise an improvement in the first precipitation stage, the precipitation stage of the modified fraction II + III and the suspension filtration stage of the modified fraction II + III.

In another embodiment, the process may comprise an improvement in the first precipitation stage, the dissolution stage of the modified fraction II + III and the suspension filtration stage of the modified fraction II + III.

In another embodiment, the process may comprise an improvement in the precipitation stage of the modified fraction II + III, the dissolution stage of the modified fraction II + III and the suspension filtration step of the modified fraction II + III .

In another embodiment, the process may comprise an improvement in the entire first stage of precipitation, the precipitation stage of the modified fraction II + III, the dissolution stage of the modified fraction II + III and the stage of suspension filtration of fraction II + III modified.

In certain embodiments, an improvement of the process in the IgG purification procedures provided herein comprises the addition by spraying of one or more solutions that would otherwise be introduced into a plasma fraction by the addition of a fluid. For example, in certain embodiments, the process improvement comprises the addition of alcohol (eg, ethanol) in a plasma fraction for the purposes of precipitation of one or more protein species by spraying. In other embodiments, solutions that can be added to a plasma fraction by spraying include, without limitation, a pH modifying solution, a solvent solution, a detergent solution, a dilution buffer, a solution modifying solution. conductivity, and the like. In a preferred embodiment, one or more precipitation steps are performed by adding alcohol to a plasma fraction by spraying. In a second preferred embodiment, one or more pH adjustment steps are performed by adding a pH modifying solution to a plasma fraction by spraying.

 twenty

In certain embodiments, another process improvement, which can be combined with any other process improvement, comprises adjusting the pH of a plasma fraction that precipitates after and / or concomitant with the addition of the precipitation agent (by example, alcohol or polyethylene glycol). In some embodiments, an improvement of the process is provided in which the pH of an actively precipitating plasma fraction is maintained throughout the entire precipitation or retention incubation stage by continuous monitoring and pH adjustment. In preferred embodiments, the pH adjustment is performed by the spray addition of a pH modifying solution.

In other embodiments, another process improvement, which can be combined with any other process improvement, comprises the use of a finely divided silica treatment step to remove impurities. 30

1. Preparation of plasma devoid of cryoprecipitate

The starting material used for the preparation of concentrated IgG compositions consists generally of recovered plasma (i.e., plasma that has been separated from whole blood ex vivo) or source plasma (i.e., plasma collected by plasmapheresis ). Typically, the purification procedure begins by defrosting the previously frozen combined plasmas, which has already been tested for safety and quality considerations. Typically, defrosting is carried out at a temperature not exceeding 6 ° C. After a complete defrosting of the frozen plasma at low temperature, cold centrifugation is performed (for example, ≤6 ° C) to separate the solid cryoprecipitates from the liquid supernatant. Alternatively, the separation step can be performed by filtration instead of centrifugation. The liquid supernatant (also called "cryoprecipitate-free plasma", after cold insoluble proteins are removed by centrifugation of freshly thawed plasma) is then processed in the next step. Several additional steps can be taken at this time for the isolation of the inhibitory bypass activity of factor eight (FEIBA), factor IX complex, factor Vll concentrate, or antithrombin III complex. Four. Five

2. First precipitation event - Section I modified

In this step, typically, the plasma devoid of cryoprecipitate is cooled to about 0 ± 1 ° C and the pH is adjusted to between about 7.0 and about 7.5, preferably between about 7.1 and 50 about 7.3, more preferably at about 7.2. In one embodiment, the pH of the plasma devoid of cryoprecipitate is adjusted to a pH of or about 7.2. Next, the precooled ethanol is added while the plasma is stirred to a target concentration of ethanol at about 8% v / v. At the same time, the temperature is further reduced to between about -4 and about 0 ° C. In a preferred embodiment, the temperature is reduced to or up to about -2 ° C, to precipitate contaminants such as α2-macroglobulin, β1A- and β1C-globulin, fibrinogen and Factor VIII. Typically, the precipitation event will include a retention time of at least about 1 hour, although shorter or longer retention times can also be employed. Subsequently, the supernatant (supernatant I) is then collected, which ideally contains all of the IgG content present in the plasma devoid of cryoprecipitate, by centrifugation, filtration, or other suitable procedure.

Compared to conventional procedures used as the first fractionation stage for plasma devoid of cryoprecipitate (Cohn et al., Supra; Oncley et al., Supra), the present invention provides, in various embodiments, procedures that result in improvement in IgG yields in the 65 fraction I supernatant. In one embodiment, the improvement in IgG performance is achieved by adding the

Spray alcohol In another embodiment, the improvement in IgG performance is achieved by adding a pH modifying agent by spraying. In yet another embodiment, the improvement in IgG performance is achieved by adjusting the pH of the solution after the alcohol addition. In a related embodiment, the improvement in IgG performance is achieved by adjusting the pH of the solution during the alcohol addition.

 5

In a specific aspect, the improvement refers to a process in which a reduced amount of IgG is lost in the precipitate fraction of the first precipitation stage. For example, in certain embodiments, a reduced amount of IgG is lost in the precipitate fraction of the first precipitation stage compared to the amount of IgG lost in the first precipitation stage of the Cohn method 6 protocol.

 10

In certain embodiments, the process improvement is produced by adjusting the pH of the solution to between about 7.0 and about 7.5 after the addition of the alcohol in precipitation. In other embodiments, the pH of the solution is adjusted to between about 7.1 and about 7.3 after the addition of the alcohol in precipitation. In still other embodiments, the pH of the solution is adjusted to about 7.0 or about 7.1, 7.2, 7.3, 7.4, or 7.5 after the addition of the alcohol in precipitation. In a particular embodiment, the pH of the solution is adjusted to approximately 7.2 after the addition of the alcohol in precipitation. As such, in certain embodiments, a reduced amount of IgG is lost in the precipitate fraction of the first precipitation stage compared to an analogous precipitation stage in which the pH of the solution is adjusted before but not after the addition of alcohol in precipitation. In one embodiment, the pH is maintained at the desired pH during the retention or incubation time of the precipitation by continuously adjusting the pH of the solution. In one embodiment, the alcohol is ethanol.

In other specific embodiments, the improvement of the process occurs by adding the alcohol in precipitation and / or the solution used to adjust the pH by spraying, rather than by the addition of a fluid. As such, in certain embodiments, a reduced amount of IgG is lost in the precipitate fraction of the first precipitation stage compared to an analogous precipitation stage in which the alcohol and / or solution used to adjust is introduced the pH by the addition of a fluid. In one embodiment, the alcohol is ethanol.

Even in other specific embodiments, the improvement occurs by adjusting the pH of the solution to between about 7.0 and about 7.5. In a preferred embodiment, the pH of solution 30 is adjusted to between about 7.1 and about 7.3. In other embodiments, the pH of the solution is adjusted to or about 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 after the addition of the alcohol in precipitation and by adding the alcohol in precipitation and / or the solution used to adjust the pH by spraying, instead of by adding fluids. In a particular embodiment, the pH of the solution is adjusted to or up to approximately 7.2 after the addition of the alcohol in precipitation and adding the alcohol in precipitation and / or the solution used to adjust the pH by spraying, in instead of by the addition of a fluid. In one embodiment, the alcohol is ethanol.

3. Second precipitation event - Section II + III modified

To further enrich the IgG content and the purity of the fractionation, the supernatant I is subjected to a second precipitation stage, which is a fractionation of the modified Cohn-Oncley fraction II + III. In general, the pH of the solution is adjusted to a pH between about 6.6 and about 6.8. In a preferred embodiment, the pH of the solution is adjusted to or about 6.7. Then, alcohol, preferably ethanol, is added to the solution while stirring to a final concentration of between about 20% and about 25% (v / v) to precipitate the IgG in the fraction. In a preferred embodiment, alcohol is added to a final concentration of one or about 25% (v / v) to precipitate the IgG in the fraction. In general, contaminants such as α1-lipoprotein, α1-antitrypsin, Gc-globulins, α1x-glycoprotein, haptoglobulin, ceruloplasmin, transferrin, hemopexin, a fraction of the Christmas factor, thyroxine-binding globulin, cholinesterase, hypertensinogen, and albumin They will not rush through these conditions. fifty

Before or concomitant with the addition of alcohol, the solution is further cooled to between about -7 ° C and about -9 ° C. In a preferred embodiment, the solution is cooled to a temperature of or about -7 ° C. After the completion of the alcohol addition, the pH of the solution is immediately adjusted to between about 6.8 and about 7.0. In a preferred embodiment, the pH of the solution is adjusted to or up to about 6.9. Typically, the precipitation event will include a retention time of at least about 10 hours, although shorter or longer retention times can also be employed. Subsequently, the precipitate (Fraction II + III modified), which ideally contains at least about 85%, preferably at least about 90%, more preferably at least about 95%, of the IgG content present in the plasma devoid of cryoprecipitate , is separated from the supernatant by centrifugation, filtration, or other suitable procedure and collected. Compared to conventional procedures employed as a second fractionation stage for plasma devoid of cryoprecipitate (Cohn et al., Supra; Oncley et al., Supra), the present invention provides, in various embodiments, procedures that result in a improvement in IgG yields in the precipitate of the modified fraction II + III. In a related embodiment, the present invention provides methods 65 that result in a reduced loss of IgG in the modified II + III supernatant.

Compared to conventional procedures employed as a second fractionation stage for plasma devoid of cryoprecipitate (Cohn et al., Supra; Oncley et al., Supra), the present invention provides, in various embodiments, procedures that result in a improvement in IgG yields in the precipitate of the modified fraction II + III. In one embodiment, the improvement is produced by the addition of alcohol by spraying. In another embodiment, the improvement is produced by the addition of a pH modifying agent by spraying. In another embodiment, the improvement is produced by adjusting the pH of the solution after the alcohol is added. In a related embodiment, the improvement is produced by adjusting the pH of the solution during the alcohol addition. In another embodiment, the improvement is produced by increasing alcohol concentration (eg, ethanol) to about 25% (v / v). In another embodiment, the improvement is produced by lowering the temperature of the precipitation stage to between about -7 ° C and -9 ° C. In a preferred embodiment, the improvement is produced by increasing the concentration of alcohol (for example, ethanol) to about 25% (v / v) and decreasing the temperature to between about -7 ° C and -9 ° C. In comparison, both Cohn et al. as Oncley et al. they perform precipitation at -5 ° C and Oncley et al. use 20% alcohol, to reduce the level of contaminants in the precipitate. Advantageously, the procedures provided herein allow maximum IgG yield without high levels of contamination in the final product. fifteen

It has been found that when the pH of the solution is adjusted to a pH of about 6.9 before the addition of the alcohol in precipitation, the pH of the solution shifts from 6.9 to between about 7.4 and about 7, 7, due in part to protein precipitation (see Figure 8). As the pH of the solution moves away from 6.9, the precipitation of IgG becomes less favorable and the precipitation of certain contaminants becomes more favorable. Advantageously, the inventors have discovered that by adjusting the pH of the solution after the addition of alcohol in precipitation, a higher percentage of IgG is recovered in the precipitate of fraction II + III.

Consequently, in one aspect, the improvement refers to a process in which a reduced amount of IgG is lost in the supernatant fraction of the precipitation stage of the modified fraction II + III. In other words, an increase in the percentage of the starting IgG is present in the precipitate of fraction II + III. In certain embodiments, the process improvement is produced by adjusting the pH of the solution to between about 6.7 and about 7.1 immediately after or during the addition of the alcohol in precipitation. In another embodiment, the improvement of the process is produced by maintaining the pH of the solution up to between about 6.7 and about 7.1 continuously during the incubation period of precipitation. In other embodiments, the pH of the solution is adjusted to between about 6.8 and about 7.0 immediately after or during the addition of the alcohol in precipitation, or to a pH of about 6.7, 6.8, 6.9, 7.0, or 7.1 immediately after or during the addition of alcohol in precipitation. In a particular embodiment, the pH of the solution is adjusted to approximately 6.9 immediately after or during the addition of the alcohol in precipitation. In certain embodiments, the pH of the solution is maintained at between about 6.8 to about 7.0 continuously during the precipitation incubation period, or at a pH of about 6.9 continuously during the period of precipitation incubation. As such, in certain embodiments, a reduced amount of IgG is lost in the supernatant fraction of the second precipitation stage compared to an analogous precipitation stage in which the pH of the solution is adjusted before but not after the addition of alcohol in precipitation or with a similar precipitation step in which the pH of the solution is not maintained during the entire precipitation incubation period. In one embodiment, the pH is maintained at the desired pH during the retention or incubation time of the precipitation by continuously adjusting the pH of the solution. In one embodiment, the alcohol is ethanol.

 Four. Five

In another embodiment, the improvement of the process is produced by adding the alcohol in precipitation and / or the solution used to adjust the pH by spraying, rather than by the addition of a fluid. As such, in certain embodiments, a reduced amount of IgG is lost in the supernatant fraction of the second precipitation stage compared to an analogous precipitation stage into which the alcohol and / or solution used to adjust the pH by addition of flux. In one embodiment, the alcohol is ethanol. fifty

In another embodiment, the improvement of the process occurs by performing the precipitation step at a temperature between about -7 ° C and about -9 ° C. In one embodiment, the precipitation step is performed at a temperature of or about -7 ° C. In another embodiment, the precipitation step is performed at a temperature of or about -8 ° C. In another embodiment, the precipitation step is performed at a temperature of or about -9 ° C. In certain embodiments, the alcohol concentration of the precipitation stage is between about 23% and about 27%. In a preferred embodiment, the alcohol concentration is between about 24% and about 26%. In another preferred embodiment, the alcohol concentration is one or about 25%. In other embodiments, the alcohol concentration may be at about 60% by 23%, 24%, 25%, 26%, or 27%. In a particular embodiment, the second precipitation stage is carried out at a temperature of or about -7 ° C with an alcohol concentration of or about 25%. In one embodiment, the alcohol is ethanol.

The effect of the increase in alcohol concentration of the second precipitation of 20%, used in Oncley et al., 65 above, to 25% and the decrease in the incubation temperature of -5 ° C, used in the procedures from Cohn and

Oncley, at or about -7 ° C is an increase of 5% to 6% in the IgG content of the precipitate of the modified fraction II + III.

In another embodiment, the process improvement is produced by adjusting the pH of the solution to between about 6.7 and about 7.1, preferably at or about 6.9, immediately after 5 or during the addition of the alcohol in precipitation. , maintaining the pH of the solution at a pH between about 6.7 and about 7.1, preferably at or about 6.9, continuously adjusting the pH during the precipitation incubation period, and adding the alcohol in precipitation and / or the solution used to adjust the pH by spraying, instead of by adding flux. In another particular embodiment, the process improvement occurs by performing the precipitation step at a temperature between about 10 -7 ° C and about -9 ° C, preferably at or about -7 ° C and precipitating the IgG with an alcohol concentration of between about 23% and about 27%, preferably at one or about 25%. In yet another particular embodiment, the process improvement is produced by incorporating all the improvements of the modified fraction II + III provided above. In a preferred embodiment, the process improvement is produced by precipitating IgG at a temperature of or about -7 ° C with ethanol at or about 25% added by spraying and then adjusting the pH of the solution to or about up to 6.9 after the addition of alcohol in precipitation. In yet another preferred embodiment, the pH of the solution is maintained at or about 6.9 during the entire incubation or precipitation retention period.

 twenty

4. Extraction of the precipitate from modified fraction II + III

To solubilize the IgG content of the precipitate of the modified fraction II + III, a cold extraction buffer is used to resuspend the precipitate of fractionation II + III in a typical proportion of 1 part of the precipitate with respect to 15 parts of the buffer of extraction. Other suitable resuspension ratios may be used, for example from about 1: 8 to about 1:30, or from about 1:10 to about 1:20, or from about 1:12 to about 1:18, or from about 1:13 to approximately 1:17, or from approximately 1:14 to approximately 1:16. In certain embodiments, the resuspension ratio may be approximately 1: 8, 1: 9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1: 29, 1:30, or greater. 30

In general, suitable solutions for the extraction of the modified II + III precipitate will have a pH between about 4.0 and about 5.5. In certain embodiments, the solution will have a pH between about 4.5 and about 5.0, in other embodiments, the extraction solution will have a pH of about 4.0, 4.1, 4.2, 4 , 3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5 . In a preferred embodiment, the pH of the extraction buffer will be about 4.5. In another preferred embodiment, the pH of the extraction buffer will be about 4.7. In another preferred embodiment, the pH of the extraction buffer will be about 4.9. In general, these pH requirements can be met using a buffering agent selected from, for example, acetate, citrate, monobasic phosphate, dibasic phosphate, mixtures thereof, and the like. Typically, suitable buffer concentrations range from about 5 to about 100 mM, or from about 10 to about 50 mM, or about about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 , 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mM.

Preferably, the extraction buffer will have a conductivity of from about 0.5 mS · cm-1 to about 2.0 mS · cm-1. For example, in certain embodiments, the conductivity of the extraction buffer will be about 0.5 mS · cm -1, or about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 mS · cm-1. One skilled in the art will know how to generate extraction buffers that have an appropriate conductivity.

In a particular embodiment, an example extraction buffer may contain 5 mM or about 5 mM monobasic sodium phosphate 50 and 5 mM acetate or about 5 mM at a pH of or about 4.5 ± 0.2 and conductivity of or about 0.7 to 0.9 mS / cm.

In general, the extraction is performed at between about 0 ° C and about 10 ° C, or between about 2 ° C and about 8 ° C. In certain embodiments, the extraction can be performed at about 0 ° C, 1 ° C, 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, or 10 ° C. In a particular embodiment, the extraction is performed at between about 2 ° C and about 10 ° C. Typically, the extraction procedure will continue for approximately 60 to approximately 300 minutes, or for approximately 120 to 240 minutes, or for approximately 150 to 210 minutes, while the suspension is continuously stirred. In certain embodiments, the extraction procedure 60 will continue for approximately 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or approximately 300 minutes. In a preferred embodiment, the extraction procedure will continue for at least 160 minutes with continuous stirring.

It has been found that using an extraction buffer containing 5 mM monobasic sodium phosphate, 5 mM acetate 65, and glacial acetic acid of 0.051% to 0.06% (v / v), a substantial increase in he

increased yield in the final IgG composition without compromising the purity of the final product. The correlation of the amount of acetic acid and the pH of the extraction buffer is shown in Figure 9. In a preferred embodiment, the precipitate of fraction II + III is extracted with a proportion of paste with respect to buffer of or about 1:15 at a pH of or about 4.5 ± 0.2.

 5

Advantageously, it has been found that compared to the current manufacturing process for GAMMAGARD® LIQUID (Baxter Healthcare), which employs an extraction buffer containing 5 mM monobasic sodium phosphate, 5 mM acetate, and 0.051 glacial acetic acid % (v / v), which by increasing the content of glacial acetic acid to about or about 0.06% (v / v), a substantial increase in yield increase in the final IgG composition can be obtained. In comparison with procedures previously used for the extraction of the precipitate formed by the second precipitation stage (GAMMAGARD® LIQUID), the present invention provides, in various embodiments, procedures that result in improved IgG yields in the suspension of the fraction II + III modified.

In one aspect, the improvement refers to a process in which a reduced amount of IgG is lost in the unsolubilized fraction of the precipitate of the modified fraction II + III. In one embodiment, the process improvement is produced by extracting the precipitate of the modified fraction II + III in a ratio of 1:15 (precipitate with respect to the buffer) with a solution containing 5 mM monobasic sodium phosphate, acetate 5 mM, and 0.06% (v / v) glacial acetic acid. In another embodiment, the improvement is produced by maintaining the pH of the solution for the duration of the extraction procedure. In one embodiment, the pH of the solution is maintained at between about 4.1 and about 4.9 for the duration of the extraction procedure. In a preferred embodiment, the pH of the solution is maintained at between about 4.2 and about 4.8 for the duration of the extraction procedure. In a more preferred embodiment, the pH of the solution is maintained at between about 4.3 and about 4.7 for the duration of the extraction procedure. In another preferred embodiment, the pH of the solution is maintained at between about 4.4 and about 4.6 for the duration of the extraction procedure. In yet another preferred embodiment, the pH of the solution is maintained at or about 4.5 for the duration of the extraction procedure.

In another aspect, the improvement refers to a process in which an increased amount of IgG of the precipitate of fraction II + III is solubilized in the stage of dissolution of fraction II + III. In one embodiment, the process improvement is produced by solubilizing the precipitate of fraction II + III in a solution buffer containing 600 ml of glacial acetic acid per 1000 l. In another embodiment, the improvement refers to a process in which impurities are reduced after IgG is solubilized in the precipitate of fraction II + III. In one embodiment, the process improvement is produced by mixing finely divided silicon dioxide (SiO2) with the suspension of fraction II + III for at least about 30 minutes.

5. Pretreatment and filtration of the suspension of fraction II + III modified

To remove the non-solubilized fraction from the precipitate of the modified fraction II + III (ie, the filter cake of the modified fraction II + III), the suspension is filtered, typically using depth filtration. Depth filters that can be used in the procedures provided herein include metallic, glass, ceramic, organic (such as diatomaceous earth) depth filters and the like. Examples of suitable filters include, without limitation, Cuno 50SA, Cuno 90SA, and Cuno VR06 (Cuno) filters. Alternatively, the separation step can be performed by centrifugation instead of filtration. Four. Five

Although the improvements in the manufacturing process described above minimize IgG losses in the initial stages of the purification process, critical impurities, including PKA activity, amidolytic activity, and fibrinogen content, are much greater when, for example, it is extracted. Paste II + III at pH 4.5 or 4.6, compared to when extraction occurs at a pH around 4.9 to 5.0 (see Examples 2 to 5). fifty

To counteract the impurities extracted in the procedures provided herein, it has been found that the purity of the IgG composition can be greatly enhanced by the addition of a pretreatment stage prior to filtration / centrifugation. In one embodiment, this pretreatment step comprises the addition of finely divided silica dioxide particles (eg, fumed silica, Aerosil® 55) followed by an incubation period of 40 to 80 minutes during which it is mixed. constant form the suspension. In certain embodiments, the incubation period will be between about 50 minutes and about 70 minutes, or about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more minutes. In general, the treatment will be carried out at between about 0 ° C and about 10 ° C, or between about 2 ° C and about 8 ° C. In certain embodiments, the treatment can be performed at about 0 ° C, 1 ° C, 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, or 10 ° C. In a particular embodiment, the treatment is carried out at between about 2 ° C and about 10 ° C.

The effect of the combustion silica treatment is exemplified by the results found in Example 17. In this example, a precipitate of fraction II + III is suspended and divided into two samples, of which one is cleared with 65 adjuvant of filter only before filtration (figure 7A) and of which one is treated with flue silica before

the addition of the filter aid and filtration (Figure 7B). As can be seen in the chromatographs and quantified data, the filtrate sample pretreated with combustion silica had a much higher IgG purity than the sample treated only with filter aid (68.8% vs. 55.7%; compare tables 17 and 18, respectively).

 5

In certain embodiments, combustion silica is added at a concentration of between about 20 g / kg paste II + III and about 100 g / kg paste II + III (ie, for a precipitate of the modified fraction II + III which extracted in a ratio of 1:15, flue silica should be added at a concentration of approximately 20 g / 16 kg suspension II + III to approximately 100 g / 16 kg suspension II + III, or at a final concentration of approximately 0.125 % (w / w) to about 0.625% (w / w)). In certain 10 embodiments, the fumed silica can be added at a concentration of about 20 g / kg paste II + III, or about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g / kg paste II + III. In a specific embodiment, fumed silica (for example, Aerosil 380 or equivalent) is added to the suspension of the modified fraction II + III to a final concentration of approximately 40 g / 16 kg / kg II + III. The mixing takes place at approximately 2 to 8 ° C for at least 50 to 70 minutes. fifteen

In certain embodiments, filter aid, for example Celpure C300 (Celpure) or Hyflo-Supper-Cel (World Minerals), will be added after treatment with silica dioxide to facilitate deep filtration. Filter aid can be added at a final concentration of from about 0.1 kg / kg paste II + III to about 0.07 kg / kg paste II + III, or from about 0.2 kg / kg paste II + III a 20 about 0.06 kg / kg paste II + III, or from about 0.3 kg / kg paste II + III to about 0.05 kg / kg paste II + III. In certain embodiments, the filter aid will be added at a final concentration of approximately 0.1 kg / kg II + III paste, or approximately 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 kg / kg II + III paste.

A significant fraction of IgG was lost during the filtration stage of the manufacturing process GAMMAGARD® LIQUID. It was found that the current post-filtration washing procedures, using 1.8 dead volumes of suspension buffer to purge the racks and filter-press lines, were insufficient for maximum IgG recovery at this stage. Surprisingly, it was found that at least 3.0 dead volumes, preferably 3.6 dead volumes, of the suspension buffer were required for the effective recovery of total IgG in the cleared suspension of the modified fraction II + III (see example 12 and figure 30 1). In certain embodiments, the filter press can be washed with any suitable suspension buffer. In a particular embodiment, the wash buffer will comprise, for example, 5 mM monobasic sodium phosphate, 5 mM acetate, and 0.015% (v / v) glacial acetic acid.

In one aspect, the improvement refers to a process in which a reduced amount of IgG is lost during the filtration step of the suspension of fraction II + III. In one embodiment, the process improvement is produced by post-washing the filter with at least about 3.6 dead volumes of solution buffer containing 150 ml of glacial acetic acid per 1000 l. The relationship between the amount of glacial acetic acid and the pH in the post-wash buffer is shown in Figure 10. In one embodiment, the pH of the post-wash extraction buffer is between about 4.6 and about 5.3. In a preferred embodiment, the pH of the post-wash buffer is between about 4.7 and about 5.2. In another preferred embodiment, the pH of the post-wash buffer is between about 4.8 and about 5.1. In yet another preferred embodiment, the pH of the post-wash buffer is between about 4.9 and about 5.0.

In comparison with the procedures previously used for the rinsing of the suspension formed from the second precipitation stage (GAMMAGARD® LIQUID), the present invention provides, in several embodiments, procedures that result in an improvement in yields of IgG and the purity in the fraction II + III suspension clarified. In one aspect, the improvement refers to a process in which a reduced amount of IgG is lost in the filter cake of the modified fraction II + III. In another aspect, the improvement refers to a process in which a reduced amount of an impurity is found in the suspension of the clarified fraction II + III 50.

In one embodiment, the process improvements are produced by the inclusion of a combustion silica treatment prior to filtration or centrifugal rinsing of a modified fraction II + III suspension. In certain embodiments, the combustion silica treatment will include the addition of from about 0.1 kg / kg paste II + III to about 0.07 kg / kg paste II + III, or about 0.2 kg / kg paste II + III at about 0.06 kg / kg paste II + III, or about 0.3 kg / kg paste II + III at about 0.05 kg / kg paste II + III, or about 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 kg / kg paste II + III, and the mixture will be incubated for between about 50 minutes and about 70 minutes, or about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more minutes at a temperature between about 2 ° C and about 8 ° C. In another embodiment, the process improvements occur by including a combustion silica treatment that reduced the levels of residual fibrinogen, amidolytic activity, and / or activity of the precalicrein activator.

In another embodiment, the process improvements are produced by washing the depth filter with between about 3 and about 5 volumes of the dead volume of the filter after completing the step of

filtration of the modified fraction II + III suspension. In certain embodiments, the filter will be washed with between about 3.5 volumes and about 4.5 volumes, or at least about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 volumes of the dead volume of the filter. In a particular embodiment, the filter press will be washed with at least about 3.6 dead volumes of the suspension buffer. 5

6. Detergent treatment

To remove additional contaminants from the filtrate of the modified fraction II + III, the sample is then subjected to a detergent treatment. Methods for detergent treatment of plasma-derived fractions 10 are well known in the art. In general, any standard non-ionic detergent treatment may be used in conjunction with the procedures provided herein. For example, an example protocol for a detergent treatment is provided below.

In summary, polysorbate-80 is added to the filtrate of the modified fraction II + III at a final concentration of approximately 0.2% (w / v) with stirring and the sample is incubated for at least 30 minutes at a temperature between approximately 2 to 8 ° C. Then, the dehydrated sodium citrate in the solution is mixed at a final concentration of about 8 g / l and the sample is incubated for an additional 30 minutes, with continuous stirring at a temperature between about 2 to 8 ° C.

 twenty

In certain embodiments, any suitable non-ionic detergent can be used. Examples of suitable non-ionic detergents include, without limitation, octylglucoside, digitonin, C12E8, Lubrol, Triton X-100, Nonidet P-40, Tween-20 (i.e., polysorbate-20), Tween-80 (i.e., polysorbate -80), an alkyl poly (ethylene oxide), a Brij detergent, a poIi (ethylene oxide) of alkylphenol, a poloxamer, octylglucoside, decylmaltoside, and the like. 25

In one embodiment, an improvement of the process is produced by adding detergent reagents (for example, polysorbate-80 and dehydrated sodium citrate) by spraying instead of by adding fluid. In other embodiments, detergent reagents can be added as solids to the filtrate of the modified fraction II + III while mixing the sample to ensure rapid distribution of the additives. In certain embodiments, it is preferable to add solid reagents by spraying the solids over a delocalized surface area of the filtrate so that local overconcentration does not occur, such as in the addition of fluid.

7. Third precipitation event - Precipitation G 35

To remove several small residual proteins, such as albumin and transferrin, a third precipitation is performed at a concentration of 25% alcohol. In summary, the pH of the detergent-treated filtrate II + III is adjusted to between about 6.8 and 7.2, preferably between about 6.9 and about 7.1, most preferably about 7.0 with a modifying solution of Suitable pH (for example, 1 M sodium hydroxide or 1 M acetic acid). Then, cold alcohol is added to the solution to a final concentration of about 25% (v / v) and the mixture is incubated while stirring at between about -6 ° C to about -10 ° C for at least 1 hour to form a third precipitate (ie precipitate G). In one embodiment, the mixture is incubated for at least 2 hours, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, or more hours. In a preferred embodiment, the mixture is incubated for at least 2 hours. In a more preferred embodiment, the mixture is incubated for at least 4 hours. In an even more preferred embodiment, the mixture is incubated for at least 8 hours.

In one aspect, an improvement of the process refers to a process in which a reduced amount of IgG is lost in the supernatant fraction of the third precipitation stage. In certain embodiments, the process improvement is produced by adjusting the pH of the solution to between about 6.8 and about 7.2 immediately after or during the addition of the alcohol in precipitation. In another embodiment, the improvement of the process occurs by maintaining the pH of the solution up to between about 6.8 and about 7.2 continuously during the precipitation incubation period. In other embodiments, the pH of the solution is adjusted to between about 6.9 and about 7.1 immediately after or during the addition of the alcohol in precipitation, or to a pH of about 6.8, 6.9 , 7.0, 7.1, or 7.2 immediately after or during the addition of the alcohol in precipitation. In a particular embodiment, the pH of the solution is adjusted to approximately 7.0 immediately after or during the addition of the alcohol in precipitation. In certain embodiments, the pH of the solution is maintained at between about 6.9 to about 7.1 continuously during the precipitation incubation period, or at a pH of about 60 approximately 7.0 continuously during the period of precipitation incubation. As such, in certain embodiments, a reduced amount of IgG is lost in the supernatant fraction of the third precipitation stage compared to an analogous precipitation stage in which the pH of the solution is adjusted before but not after the addition of alcohol in precipitation or with an analogous precipitation stage in which the pH of the solution is not maintained during the entire precipitation incubation period. In a mode of 65

embodiment, the pH is maintained at the desired pH during the retention or incubation time of the precipitation by continuously adjusting the pH of the solution. In one embodiment, the alcohol is ethanol.

In another embodiment, the improvement of the process is produced by adding the alcohol in precipitation and / or the solution used to adjust the pH by spraying, rather than by the addition of a fluid. As such, in certain 5 embodiments, a reduced amount of IgG is lost in the supernatant fraction of the third precipitation stage compared to an analogous precipitation stage in which the alcohol and / or solution used to adjust is introduced. the pH by the addition of a fluid. In one embodiment, the alcohol is ethanol.

8. Suspension and filtration of precipitate G (PptG) 10

To solubilize the IgG content of precipitate G, a cold extraction buffer is used to resuspend the PptG. In summary, the precipitate G 1 to 3.5 is dissolved in injectable water (WFI) at between about 0 ° C and about 8 ° C to achieve a value of AU280-320 between about 40 to 95. The pH is then adjusted end of the solution, which is stirred for at least 2 hours, up to or approximately up to 5.2 ± 0.2. In one embodiment, this pH adjustment is made with 1 M acetic acid. To increase the solubility of the IgG, the conductivity of the suspension is increased to between about 2.5 and about 6.0 mS / cm. In one embodiment, the conductivity is increased by the addition of sodium chloride. Next, the suspended PptG solution is filtered with a suitable depth filter having a nominal pore size between about 0.1 µm and about 0.4 µm to remove any undissolved particles. In one embodiment, the nominal pore size of the depth filter is approximately 0.2 μm (for example, Cuno VR06 filter or equivalent) to obtain a clarified filtrate. In another embodiment, the suspended PptG solution is filtered to recover a clarified supernatant. Post-washing of the filter is performed using a solution of sodium chloride with a conductivity between about 2.5 and about 6.0 mS / cm. Typically, suitable solutions for the extraction of precipitate G include, WFI and buffers with low conductivity. In one embodiment, a low conductivity buffer has a conductivity of less than about 10 mS / cm. In a preferred embodiment, the low conductivity buffer has a conductivity of less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mS / cm. In a preferred embodiment, the low conductivity buffer has a conductivity of less than about 6 mS / cm. In another preferred embodiment, the low conductivity buffer has a conductivity of less than about 4 mS / cm. In another preferred embodiment, the low conductivity buffer has a conductivity of less than about 2 mS / cm.

9. Solvent-detergent treatment

 35

To inactivate various viral contaminants that may be present in plasma-derived products, the clarified PptG filtrate is then subjected to a solvent-detergent (S / D) treatment. Methods for detergent treatment of plasma-derived fractions are well known in the art (for review see, Pelletier JP et al., Best Pract Res Clin Haematol. 2006; 19 (1): 205-42). In general, any standard S / D treatment may be used in conjunction with the procedures provided herein. For example, an example protocol for an S / D treatment is provided below.

In summary, Triton X-100, Tween-20, and tri (n-butyl) phosphate (TNBP) are added to the clarified PptG filtrate at final concentrations of approximately 1.0%, 0.3%, and 0, 3%, respectively. Then, the mixture is stirred at a temperature between about 18 ° C and about 25 ° C for at least 45 an hour.

In one embodiment, an improvement of the process is produced by adding the S / D reagents (for example, Triton X-100, Tween-20, and TNBP) by spraying instead of by adding fluid. In other embodiments, detergent reagents as solids can be added to the clarified PptG filtrate, which is being mixed to ensure rapid distribution of the S / D components. In certain embodiments, it is preferable to add solid reagents by spraying the solids over a delocalized surface area of the filtrate so that local overconcentration does not occur, such as in the addition of fluid.

10. Ion exchange chromatography 55

To further purify and concentrate the IgG of the S / D treated PptG filtrate, cation exchange and / or anion exchange chromatography can be used. Methods for purifying and concentrating IgG using ion exchange chromatography are well known in the art. For example, US Pat. UU. No. 5,886,154 describes a process in which a precipitate of fraction II + III is extracted at low pH (between approximately 60 and 3.8 and 4.5), followed by precipitation of IgG using caprylic acid, and finally the Two-stage implementation of anion exchange chromatography. U.S. Patent UU. No. 6,069,236 describes a chromatographic IgG purification scheme that is not in any way based on the precipitation of alcohol. PCT Publication No. WO 2005/073252 describes an IgG purification procedure that involves the extraction of a precipitate of fraction II + III, treatment with caprylic acid, treatment with PEG, and a single stage of anion exchange chromatography. . U.S. Patent UU. No. 7,186,410 describes an IgG purification procedure that involves the

extraction of a precipitate from fraction I + II + III or fraction II followed by a single anion exchange stage performed at alkaline pH. U.S. Patent UU. No. 7,553,938 describes a procedure that involves the extraction of a precipitate of fraction I + II + III or fraction II + III, treatment with caprylate, and one or two stages of anion exchange chromatography. U.S. Patent UU. No. 6,093,324 describes a purification process comprising the use of a macroporous anion exchange resin that is operated at a pH of between about 6.0 and about 6.6. U.S. Patent UU. No. 6,835,379 describes a purification procedure that is based on cation exchange chromatography in the absence of alcohol fractionation. The disclosures of previous publications are incorporated herein by reference in their entirety for all purposes.

 10

In one embodiment of the methods of the present invention, the PptG filtrate treated with S / D can be subjected to both cation exchange chromatography and anion exchange chromatography. For example, in one embodiment, the PptG filtrate treated with S / D is passed through a cation exchange column, which binds the IgG in the solution. The S / D reagents can then be removed by washing the absorbed IgG, which is subsequently separated by elution from the column with a high pH elution buffer having a pH between about 8.0 and 9.0 . In this way, the cation exchange chromatography step can be used to remove the S / D reagents from the preparation, to concentrate the solution containing the IgG, or both. In certain embodiments, the pH elution buffer may have a pH of between about 8.2 and about 8.8, or between about 8.4 and about 8.6, or a pH of about 8.0, 8 , 1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In a preferred embodiment, the pH of the elution buffer is approximately 8.5 ± 0.1.

In certain embodiments, the elution of the cation exchange column can be adjusted to a lower pH, for example between about 5.5 and about 6.5, and diluted with an appropriate buffer so as to reduce the conductivity of the solution. In certain embodiments, the pH of the cation exchange eluate 25 can be adjusted to a pH between about 5.7 and about 6.3, or between about 5.9 and about 6.1, or a pH of about 5 , 5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In a preferred embodiment, the pH of the eluate is adjusted to a pH of about 6.0 ± 0.1. Next, the eluate is loaded onto an anion exchange column, which binds several contaminants discovered in the preparation. The flow through the column, which contains the IgG fraction, is collected during loading and washing of the column. In certain embodiments, the ion exchange chromatographic steps of the present invention can be performed in column mode, in batch mode, or in a combination of the two.

In certain embodiments, an improvement of the process is produced by adding the solution used to adjust the pH by spraying, rather than by adding flux.

11. Nanofiltration and ultra / diafiltration

To further reduce the viral load of the IgG composition provided herein, the effluent of the anion exchange column can be nanofiltered using a suitable nanofiltration device. In certain embodiments, the nanofiltration device will have an average pore size between about 15 nm and about 200 nm. Examples of nanofilters suitable for this use include, without limitation, DVD, DV 50, DV 20 (Pall), Viresolve NFP, Viresolve NFR (Millipore), Planova 15N, 20N, 35N, and 75N (Planova). In a specific embodiment, the nanofilter can have an average pore size of between about 15 nm and about 72 nm, or between about 19 nm and about 35 nm, or about 15 nm, 19 nm, 35 nm, or 72 nm In a preferred embodiment, the nanofilter will have an average pore size of approximately 35 nm, such as an Asahi PLANOVA 35N filter or equivalent thereof.

Optionally, ultrafiltration / diafiltration can be performed to further concentrate the nanofiltrate. In one embodiment, an open channel membrane with a specifically designed post-wash is used and the formulation near the end of the production process causes the resulting IgG compositions to have a protein concentration approximately twice as high (200 mg / ml). ) in comparison with the IVIGs of the prior art (for example, GAMMAGARD® LIQUID) without affecting performance and storage stability. With most commercially available ultrafiltration membranes, a concentration of 200 mg / ml of IgG cannot be achieved without significant protein losses. These membranes will be blocked soon and therefore, it is difficult to achieve adequate post-wash. Therefore, open channel membrane configurations should be used. Even with open channel membranes, a post-wash procedure specifically designed to obtain the required concentration should be used without a significant loss of protein (loss less than 2%). Even more surprising is the fact that the highest protein concentration of 200 mg / ml no 60 achieves the inactivation capacity of the virus of the storage stage at low pH.

After nanofiltration, the filtrate can be further concentrated by ultrafiltration / diafiltration. In one embodiment, the nanofiltration can be concentrated by ultrafiltration to a protein concentration of between about 2% and about 10% (w / v). In certain embodiments, ultrafiltration 65 is carried out in a cassette with an open channel screen and the ultrafiltration membrane has a weight limit

Molecular Nominal (NMWCO) less than about 100 kDa or less than about 90, 80, 70, 60, 50, 40, 30 kDa, or less kDa. In a preferred embodiment, the ultrafiltration membrane has an nmWCO of no more than 50 kDa.

After completion of the ultrafiltration stage, the concentrate can be further concentrated by means of diafiltration against a solution suitable for intravenous or intramuscular administration. In certain embodiments, the diafiltration solution may comprise a stabilizing and / or buffering agent. In a preferred embodiment, the stabilizing and buffering agent is glycine in an appropriate concentration, for example between about 0.20 M and about 0.30 M, or between about 0.22 M and about 0.28 M, or between about 0.24 M and about 0.26 mM, or at a concentration of about 10, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In a preferred embodiment, the diafiltration buffer contains 0.25 M glycine or about 0.25 M.

Typically, the minimum exchange volume is at least about 3 times the volume of the original concentrate or at least about 4, 5, 6, 7, 8, 9, or more times the volume of the original concentrate. The 15 IgG solution can be concentrated to a protein concentration of about 5% and about 25% (w / v), or between about 6% and about 18% (w / v), or between about 7% and about 16% (w / v), or between about 8% and about 14% (w / v), or between about 9% and about 12%, or up to a final concentration of about 5%, or 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 20 24%, 25% or greater. In one embodiment, a final protein concentration of at least about 23% is achieved without adding the post-wash fraction to the concentrated solution. In another embodiment, a final protein concentration of at least about 24% is achieved without adding the post-wash fraction to the concentrated solution. A final protein concentration of at least about 25% is achieved without adding the post-wash fraction to the concentrated solution. Typically, at the end of the concentration procedure, the pH of the solution will be between about 4.6 to 5.1.

In an exemplary embodiment, the pH of the IgG composition is adjusted to approximately 4.5 before ultrafiltration. The solution is concentrated to a protein concentration of 5 ± 2% w / v through ultrafiltration. The UF membrane has a nominal molecular weight limit (NMWCO) of 50,000 Dalton or less 30 (Millipore Pellicon polyether sulfone membrane). The concentrate is subjected to diafiltration against ten volumes of 0.25 M glycine solution, pH 4.5 ± 0.2. Throughout the ultra-diafiltration operation, the solution is maintained at a temperature between about 2 ° C to about 8 ° C. After diafiltration, the solution is concentrated to a protein concentration of at least 11% (w / v).

 35

12. Formulation

After completion of the diafiltration stage, the protein concentration of the solution is adjusted with the diafiltration buffer to a final concentration of between about 5% and about 20% (w / v), or between about 6% and about 18% (w / v), or between about 7% and 40 about 16% (w / v), or between about 8% and about 14% (w / v), or between about 9% and approximately 12%, or up to a final concentration of approximately 5%, or 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% , 16%, 17%, 18%, 19%, or 20%. In a preferred embodiment, the final protein concentration of the solution is between about 9% and about 11%, more preferably it is about 10%. Four. Five

The formulated mass solution is further sterilized by filtering through a membrane filter with an absolute pore size of no more than about 0.22 micrometers, for example about 0.2 micrometers. Next, the solution is aseptically dispensed in final containers for proper sealing, with samples taken for sampling. fifty

In one embodiment, the IgG composition is further adjusted to a concentration of approximately 10.2 ± 0.2% (w / v) with a diafiltration buffer. The pH is adjusted to about 4.4 to about 4.9 if necessary. Finally, the solution is sterile filtered and incubated for three weeks at or about 30 ° C. 55

13. Adding alcohol

Advantageously, it has been found that, in order to fractionate the plasma IgG, the addition of alcohol by spraying instead of the addition of a flower results in a reduction in the loss of IgG yields. Without compromising with any theory, during the addition of flux to a plasma fraction, the transient local overconcentration of alcohol in the fluid access can lead to protein denaturation and irreversible loss and / or precipitation of IgG during stages in which IgG must remain in the supernatant. In addition, these effects can be amplified when large volumes of alcohol need to be added, such as industrial scale purifications that involve the fractionation of at least 100 µl of combined plasmas. 65

The effect of the addition of alcohol by means of spraying is exemplified in example 14, in which samples of plasma devoid of cryoprecipitate with 8% ethanol introduced by the addition of a fluid (1 and 2) or spray addition ( 3 and 4). As can be seen in Table 14, almost 100% of the IgG present in the plasma devoid of cryoprecipitate is recovered in the supernatant when ethanol is added to the sample by spraying, while from 4 to 5% of the IgG It is lost after the addition of alcohol flux. This results in a loss of IgG between about 0.20 and 0.25 g / l at this stage alone. In terms of production levels in 2007, this translates into a loss of approximately 5.3 million grams (5,300 kilograms) of IgG. Given the current market price for IVIG, which varies between $ 50 and $ 100 per gram, a loss of 4 to 5% at this stage represents a global economic loss of up to five hundred million dollars annually.

 10

Accordingly, in one aspect of the procedures provided herein, one or more precipitation steps are performed by the alcohol spraying. In certain embodiments, the spray addition can be performed by any pressurized device, such as a container (for example, a spray bottle), which has a spray head or a nozzle and is operated manually or automatically to generate a fine vaporization of a liquid. In certain embodiments, the spray addition is performed while the system is continuously stirred or mixed in another way to ensure a rapid and equitable distribution of the liquid within the system.

14. pH adjustment

 twenty

The protein precipitation profile of plasma fractions is highly dependent on the pH of the solution from which the plasma proteins precipitate. This fact has been exploited by scientists who fractionate plasma proteins since the introduction of the Cohn and Oncley procedures in 1946 and 1949, respectively. Traditionally, the pH of a plasma fraction is adjusted before the addition of alcohol to facilitate the highest recovery yields for the component of interest. Advantageously, it has now been discovered that adjusting the pH of the solution directly after the addition of alcohol or concomitant with the addition of alcohol results in a more defined and reproducible precipitation. It was found that the addition of ethanol to the plasma fractions results in fluctuations in the pH of the solution, in general, raising the pH of the solution. As such, when adjusting the pH of a plasma fraction to a predetermined pH before but not after the addition of alcohol, the precipitation reaction will occur at a non-optimal pH. 30

Likewise, the precipitation of proteins from a plasma fraction will affect the electrostatic environment and therefore alter the pH of the solution. Consequently, as a precipitation event is allowed to progress, the pH of the solution will begin to diverge from the predetermined pH value that allows maximum recovery of the protein species of interest. This is especially true for precipitation events in which a large fraction of the protein is to be precipitated, precipitation events in which a high alcohol content is used, and precipitation events that require a long incubation period.

The effect of adjusting the pH of a plasma fraction is exemplified by the results found in example 16. In this example, IgG was precipitated from two samples of a fraction of supernatant I after addition by alcohol spraying. The pH of both samples was adjusted to 6.7 before the alcohol addition and readjusted to 6.9 after the alcohol addition but before the 10 hour precipitation incubation step. In the first sample (reference), the pH was not adjusted during the 10-hour incubation, while the sample two (continuous adjustment), the pH was constantly adjusted to pH 6.9 during the 10-hour incubation. As can be seen in Table 16, after the removal of the precipitate from the modified fraction II + III from the samples, the first supernatant 45 contained 0.2 g IgG / l plasma, while the second sample, in which kept the pH constant during the precipitation incubation, it contained only 0.13 g IgG / l plasma. The reduction in the loss of 0.07 g IgG / l plasma in the second sample represents, in terms of production levels in 2007, a loss of approximately 1.9 million grams (1,900 kilograms) of IgG. Given the current market price for IVIG, which ranges from $ 50 to $ 100 per gram, a 1.5% loss at this stage represents a global economic loss of up to $ 200 million annually.

Accordingly, in one aspect of the procedures provided herein, the pH of a plasma fraction is adjusted directly after the addition of alcohol. In related embodiments, the pH can be adjusted before or after the alcohol addition, or during and after the alcohol addition, or before, during and after the alcohol addition. In a related embodiment, the pH of a solution is continuously adjusted during one or more incubations or alcohol precipitation events. In certain embodiments, the pH of a solution is continuously adjusted or maintained while the system is continuously stirred or otherwise mixed to ensure a rapid and equitable distribution of the pH modifying agent within the system. 60

Similar to the case of the addition of fluoro alcohol, it has now been discovered that the addition of large volume flux of a pH modifying agent can cause transient local pH variations, which results in denaturation or precipitation of unwanted protein. . Accordingly, in one embodiment of the procedures provided herein, pH modifying agents can be introduced in one or more stages of plasma fractionation by spray addition. In another embodiment of the

Procedures provided herein, the pH of a plasma or precipitation fraction step can be adjusted by spray addition of a pH modifying agent. In certain embodiments, the spray addition can be performed by any pressurized device, such as a container (for example, a spray bottle), which has a spray head or a nozzle and is operated manually or automatically to generate a fine vaporization of a liquid. In certain embodiments, the spray addition is performed while the system is continuously stirred or mixed in another way to ensure a rapid and equitable distribution of the liquid within the system.

III. Concentrated IgG Compositions

 10

IVIG compositions comprising antibody for the treatment of certain autoimmune conditions have been described. (See, for example, U.S. Patent Publications No. US 2002/0114802, US 2003/0099635 and US 2002/0098182.) The IVIG compositions disclosed in these references include polyclonal antibodies.

 fifteen

1. Aqueous IgG compositions

In one aspect, the present invention relates to aqueous IgG compositions prepared by the methods provided herein. In general, IgG compositions prepared by the novel methods described herein will have a high IgG content and purity. For example, the 20 IgG compositions provided herein may have a protein concentration of at least about 3% (w / v) and an IgG content of purity greater than about 90%. These high purity IgG compositions are suitable for therapeutic administration, for example, for IVIG treatment. In one embodiment, the concentration of IgG is approximately 10% and is used for intravenous administration. In another embodiment, the concentration is approximately 20% and 25 is used for subcutaneous or intramuscular administration.

In one embodiment, the present invention provides an aqueous IgG composition prepared by a process comprising the steps of (a) precipitating a plasma fraction devoid of cryoprecipitate, in a first precipitation stage, with alcohol between about 6% and about 10% at a pH of between about 6.7 and about 7.3 to obtain an IgG-enriched supernatant, (b) precipitate IgG from the supernatant with alcohol between about 20% and about 30% at a pH between about 6.7 and about 7.3 to form a first precipitate, (c) resuspend the first precipitate formed in step (b) to form a suspension, (d) treat the suspension formed in step (c) with a detergent, (e) precipitate IgG from the suspension with alcohol between about 20% and about 35% at a pH between about 6.7 and about 7.3 to form a second precipitate or, (f) resuspend the second precipitate formed in step (e) to form a suspension, (g) treat the suspension formed in step (f) with a solvent and / or detergent, and (h) perform at least one ion exchange chromatography fractionation thereby preparing a concentrated IgG composition.

 40

In a specific embodiment, an IgG composition is provided which is prepared by a process comprising the steps of (a) adjusting the pH of a fraction of plasma devoid of cryoprecipitate to approximately 7.0, (b) adjusting the concentration of ethanol from the plasma fraction devoid of cryoprecipitate from step (a) to about 25% (v / v) at a temperature between about -5 ° C and about -9 ° C, thereby forming a mixture, (c) separate liquid and precipitate from the mixture of step 45 (b), (d) resuspend the precipitate from step (c) with a buffer containing phosphate and acetate, in which the pH of the buffer is adjusted with 600 ml of acid glacial acetic per 1000 l of buffer, thereby forming a suspension, (e) mixing silicon dioxide (SiO2) finely divided with the suspension of step (d) for at least about 30 minutes, (f) filtering the suspension with a filter-press, forming of this way a filtrate, (g) wash the filter-press with at least 3 dead volumes of filter-press of a buffer containing phosphate and acetate, in which the pH of the buffer is adjusted with 150 ml of glacial acetic acid by 1000 l of buffer, thus forming a wash solution, (h) combine the filtrate of step (f) with the wash solution of step (g), thereby forming a solution, and treat the solution with a detergent, (i) adjust the pH of the solution of step (h) to about 7.0 and add ethanol to a final concentration of about 25%, thereby forming a precipitate, (j) separating liquid and precipitate of the mixture of step (i), (k) dissolve the precipitate in an aqueous solution comprising a solvent or detergent and hold the solution for at least 60 minutes, (1) pass the solution after the step (k ) through a cation exchange chromatography column and elute the pr oteins absorbed in the column in an eluate, (m) passing the eluate from step (1) through an anion exchange chromatography column to generate an effluent, (n) passing the effluent from stage (m) through a nanofilter to generate a nanofiltrate, (or) passing the nanofiltrate of step (n) through an ultrafiltration membrane to generate an ultrafiltrate, and (p) diafiltering the ultrafiltrate of the stage (or ) against a diafiltration buffer to generate a diafiltrate having a protein concentration between about 8% (w / v) and about 12% (w / v), thereby obtaining a concentrated IgG composition.

 65

In certain embodiments, aqueous IgG compositions are prepared using a process provided herein that comprises improvements in two or more of the steps of the fractionation process described above. For example, in certain embodiments, improvements can be found in the precipitation stage, the precipitation stage of the modified fraction II + III, the dissolution stage of the modified fraction II + III, and / or the stage of suspension filtration of fraction II + III modified. 5

In one embodiment, an aqueous IgG composition is provided which is prepared by a purification process described herein, in which the process comprises the spray addition of one or more solutions that could otherwise be introduced into a fraction of plasma by the addition of a fluid. For example, in certain embodiments, the process will comprise introducing alcohol (for example, ethanol) into a plasma fraction by spraying. In other embodiments, solutions that can be added to a plasma fraction by spraying include, without limitation, a pH modifying solution, a solvent solution, a detergent solution, a dilution buffer, a conductivity modifying solution. , and the like. In a preferred embodiment, one or more precipitation steps are performed by adding alcohol to a plasma fraction by spraying. In a second preferred embodiment, one or more pH adjustment steps are performed by adding a pH modifying solution to a plasma fraction by spraying.

In certain embodiments, an aqueous IgG composition is provided which is prepared by a purification process described herein, wherein the process comprises adjusting the pH of a plasma fraction that precipitates after and / or concomitant with the addition of the precipitating agent (for example, alcohol or polyethylene glycol). In some embodiments, an improvement of the process is provided in which the pH of an actively precipitating plasma fraction is maintained throughout the entire precipitation or retention incubation stage by continuous monitoring and pH adjustment. In preferred embodiments, the pH adjustment is performed by the spray addition of a pH modifying solution. 25

In one embodiment, the present invention provides aqueous IgG compositions comprising a protein concentration of between about 30 g / l and about 250 g / l. In certain embodiments, the protein concentration of the IgG composition is between about 50 g / l and about 200 g / l, or between about 70 g / l and about 150 g / l, or between about 30 90 g / l and about 120 g / l, or any suitable concentration within these ranges, for example about 30 g / l, or about 35 g / l, 40 g / l, 45 g / l, 50 g / l, 55 g / l, 60 g / l, 65 g / l, 70 g / l, 75 g / l, 80 g / l, 85 g / l, 90 g / l, 95 g / l, 100 g / l, 105 g / l, 110 g / l, 115 g / l, 120 g / l, 125 g / l, 130 g / l, 135 g / l, 140 g / l, 145 g / l, 150 g / l, 155 g / l, 160 g / l, 165 g / l, 170 g / l, 175 g / l, 180 g / l, 185 g / l, 190 g / l, 195 g / l, 200 g / l, 205 g / l, 210 g / l, 215 g / l, 220 g / l, 225 g / l, 230 g / l, 235 g / l, 240 g / l, 245 g / l, 250 g / l, or greater. In a preferred embodiment, the aqueous IgG composition 35 will have a concentration of one or about 10%. In a particularly preferred embodiment, the composition will have a concentration of 10.2 ± 0.2% (w / v). In another preferred embodiment, the aqueous IgG composition will have a concentration of or about 20%.

The procedures provided herein allow the preparation of IgG compositions that have very high levels of purity. In one embodiment, at least about 95% of the total protein in a composition provided herein will be IgG. In other embodiments, at least about 96% of the protein is IgG, or at least about 97%, 98%, 99%, 99.5%, or more of the total protein in the composition will be IgG. In a preferred embodiment, at least 97% of the total protein in the composition will be IgG. In another preferred embodiment, at least 98% of the total protein of the composition will be IgG. In another preferred embodiment, at least 99% of the total protein of the composition will be IgG.

Similarly, the procedures provided herein allow the preparation of IgG compositions containing extremely low levels of pollutants. For example, Table 50 19 provides the results of the impurity test for three bulk IgG solutions prepared by the improved procedures provided herein. In certain embodiments, IgG compositions containing less than about 140 mg / l IgA are provided. In other embodiments, the IgG composition will contain less than about 60 mg / l IgA, preferably less than about 40 mg / l IgA, most preferably less than about 30 mg / l IgA. 55

In another embodiment, IgG compositions containing less than about 50 mg / l IgM are provided. In other embodiments, the IgG composition will contain less than about 25 mg / l IgM, preferably less than about 10 mg / l IgM, more preferably less than about 5 mg / l IgM, more preferably less than about 4 mg / l IgM, more preferably less than about 60 mg / l IgM, most preferably less than about 2.5 mg / l IgM.

In another embodiment, IgG compositions containing less than about 100 PL-1 nmol / ml min of amidolytic activity are provided. In other embodiments, the IgG composition will contain less than about 50 PL-1 nmol / ml min of amidolytic activity, preferably less than about 25 65 PL-1 nmol / ml min of amidolytic activity, more preferably less than about 20 PL -1 nmol / ml min of

amidolytic activity, more preferably less than about 15 PL-1 nmol / ml min of amidolytic activity, most preferably less than about 10 PL-1 nmol / ml min of amidolytic activity.

In another embodiment, IgG compositions containing less than about 20 mg / l fibrinogen are provided. In other embodiments, the IgG composition will contain less than about 5-10 mg / l of fibrinogen, preferably less than about 5 mg / l of fibrinogen, more preferably less than about 2.5 mg / l of fibrinogen, more preferably less of about 1 mg / l of fibrinogen, more preferably less than about 0.5 mg / l of fibrinogen, most preferably less than about 0.25 mg / l of fibrinogen.

 10

In yet another embodiment, IgG compositions are provided consisting primarily of IgG monomers / dimers. In one embodiment, an IgG composition is provided in which at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99, 4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the IgG is monomeric or dimeric. In a preferred embodiment, an IgG composition is provided in which at least 97% of the IgG is monomeric or dimeric. In a more preferred embodiment, at least 99% of the IgG is monomeric or dimeric. In a more preferred embodiment, at least 99.5% of the IgG is monomeric or dimeric. In a more preferred embodiment, at least 99.7% of the IgG is monomeric or dimeric.

2. Pharmaceutical compositions 20

In another aspect, the present invention provides pharmaceutical formulations and compositions comprising purified IgG prepared by the procedures provided herein. In general, the formulations and pharmaceutical compositions of IgG prepared by the novel methods described herein will have a high IgG content and purity. For example, the pharmaceutical formulations and compositions of IgG provided herein may have a protein concentration of at least about 7% (w / v) and an IgG content of purity greater than about 95%. These high purity IgG pharmaceutical formulations and compositions are suitable for therapeutic administration, for example, for IVIG treatment. In a preferred embodiment, a pharmaceutical IgG composition is formulated for intravenous administration (eg, IVIG treatment). 30

In one embodiment, the pharmaceutical compositions provided herein are prepared by formulating an isolated aqueous IgG composition using a procedure provided herein. In general, the formulated composition will have been subjected to at least one, preferably at least two, most preferably at least three, stages of inactivation or viral withdrawal. Non-limiting examples of viral inactivation or withdrawal steps that can be employed with the procedures provided herein include solvent-detergent treatment (Horowitz et al., Blood Coagul Fibrinolysis 1994 (5 Suppl. 3): S21- S28 and Kreil et al., Transfusion 2003 (43): 1023-1028, of which both are expressly incorporated herein by reference in their entirety for all purposes), nanofiltration (Hamamoto et al., Vox Sang 1989 ( 56) 230-236 and Yuasa et al., J Gen Virol. 1991 (72 (pt 8)): 2021-2024, of which both are expressly incorporated herein by reference in their entirety for all purposes) , incubation at low pH at high temperatures (Kempf et al., Transfusion 1991 (31) 423-427 and Louie et al., Biologicals 1994 (22): 13-19).

In certain embodiments, pharmaceutical formulations having an IgG content of between about 80 g / l IgG and about 120 g / l IgG are provided. In general, these IVIG formulations are prepared by isolating a plasma IgG composition using a procedure described herein, concentrating the composition, and formulating the concentrated composition in a solution suitable for intravenous administration. IgG compositions can be concentrated using any suitable procedure known to one skilled in the art. In one embodiment, the composition is concentrated by ultrafiltration / diafiltration. In some embodiments, the ultrafiltration device used to concentrate the composition will employ an ultrafiltration membrane having a nominal molecular weight limit (NMWCO) of less than about 100 kDa or less than about 90, 80, 70, 60, 50 , 40, 30, or less kDa. In a preferred embodiment, the ultrafiltration membrane has an nmWCO of no more than 50 kDa. Buffer exchange can be achieved using any suitable technique known to one skilled in the art. In a specific embodiment, buffer exchange is achieved by diafiltration. 55

In a specific embodiment, a pharmaceutical composition of IgG is provided, in which the IgG composition was purified from plasma using a method comprising the steps of (a) precipitating a plasma fraction devoid of cryoprecipitate, in a first stage of precipitation, with alcohol between about 6% and about 10% at a pH of between about 6.7 and about 7.3 to obtain a supernatant enriched in IgG, (b) precipitate IgG of the supernatant with alcohol between about a 20% and about 30% at a pH between about 6.7 and about 7.3 to form a first precipitate, (c) resuspend the first precipitate formed in step (b) to form a suspension, (d) treat the suspension formed in step (c) with a detergent, (e) precipitate IgG from the suspension with alcohol between about 20% and about 30% at a pH between about 6.7 and about 65 7.3 to form a second precipitate, (f) resuspend the second precipitate formed in step (e) to form

a suspension, (g) treating the suspension formed in step (f) with a solvent and / or detergent, (h) performing at least one fractionation by ion exchange chromatography; (i) perform a solvent-detergent treatment, and (j) subject the composition to nanofiltration, thereby preparing an IgG composition.

In a specific embodiment, a pharmaceutical IgG composition is provided, in which the IgG composition 5 was purified from plasma using a method comprising the steps of (a) adjusting the pH of a plasma fraction devoid of cryoprecipitate until about 7.0, (b) adjust the ethanol concentration of the cryoprecipitated plasma fraction of step (a) to about 25% (v / v) at a temperature between about -5 ° C and about -9 ° C , thereby forming a mixture, (c) separating liquid and precipitate from the mixture of stage (b), (d) resuspending the precipitate from stage (c) with a buffer containing 10 phosphate and acetate, in which the pH of the buffer is adjusted with 600 ml of glacial acetic acid per 1000 l of buffer, thereby forming a suspension, (e) mixing silicon dioxide (SiO2) finely divided with the suspension of step (d) for at least approximately 30 minutes, (f) filter the suspension with a filter-press, thereby forming a filtrate, (g) wash the filter-press with at least 3 dead volumes of filter-press from a buffer containing phosphate and acetate, in which the pH of the buffer is adjusted with 150 ml of glacial acetic acid per 15 1000 l of buffer, thereby forming a wash solution, (h) combining the filtrate of step (f) with the washing solution of stage (g ), thereby forming a solution, and treating the solution with a detergent, (i) adjust the pH of the solution in step (h) to approximately 7.0 and add ethanol to a final concentration of approximately 25%, thereby forming a precipitate, (j) separating liquid and precipitate from the mixture of step (i), (k) dissolving the precipitate in an aqueous solution comprising a solvent or detergent and maintaining the solution for at least 60 minutes , (1) pass the solution after step (k) through a column na cation exchange chromatography and elute the absorbed proteins in the column in an eluate, (m) pass the eluate from step (1) through an anion exchange chromatography column to generate an effluent, (n) make passing the effluent from stage (m) through a nanofilter to generate a nanofiltrate, (or) passing the nanofiltrate from stage (n) through an ultrafiltration membrane to generate an ultrafiltrate, and (p) submit The ultrafiltrate of step (o) is diafiltered against a diafiltration buffer to generate a diafiltrate having a protein concentration between about 8% (w / v) and about 12% (w / v), thereby obtaining a concentrated IgG composition.

In certain embodiments, a pharmaceutical IgG composition is provided, in which the IgG composition is prepared using a method provided herein that comprises improvements in two or more of the steps of the fractionation process described above. For example, in certain embodiments, improvements can be found in the precipitation stage, the precipitation stage of the modified fraction II + III, the dissolution stage of the modified fraction II + III, and / or the stage of suspension filtration of fraction II + III modified. 35

In certain embodiments, an IgG pharmaceutical composition is provided, in which the IgG composition is prepared using a purification procedure described herein, in which the process comprises the spray addition of one or more solutions that they could otherwise be introduced into a fraction of plasma by the addition of a fluid. For example, in certain embodiments, the process will comprise the introduction of alcohol (eg, ethanol) into a plasma fraction by spraying. In other embodiments, solutions that can be added to a plasma fraction by spraying include, without limitation, a pH modifying solution, a solvent solution, a detergent solution, a dilution buffer, a conductivity modifying solution. , and the like. In a preferred embodiment, one or more precipitation steps are performed by adding alcohol to a plasma fraction by spraying. In a second preferred embodiment, one or more pH adjustment steps are performed by adding a pH modifying solution to a plasma fraction by spraying.

In certain embodiments, an IgG pharmaceutical composition is provided, in which the IgG composition is prepared by a purification procedure described herein, wherein the method comprises adjusting the pH of a plasma fraction that precipitates after and / or concomitant with the addition of the precipitating agent (for example, alcohol or polyethylene glycol). In some embodiments, an improvement of the process is provided in which the pH of an actively precipitating plasma fraction is maintained throughout the entire precipitation or retention incubation stage by continuous monitoring and pH adjustment. In preferred embodiments, the pH adjustment is made by the addition of a pH modifying solution by spraying.

In one embodiment, the present invention provides a pharmaceutical composition of IgG comprising a protein concentration of between about 70 g / l and about 130 g / l. In certain embodiments, the protein concentration of the IgG composition is between about 80 g / l and 60 about 120 g / l, preferably between about 90 g / l and about 110 g / l, most preferably about 100 g / l, or any suitable concentration within these ranges, for example approximately 70 g / l, 75 g / l, 80 g / l, 85 g / l, 90 g / l, 95 g / l, 100 g / l, 105 g / l, 110 g / l, 115 g / l, 120 g / l, 125 g / l, or 130 g / l. In a preferred embodiment, a pharmaceutical composition is provided having a protein concentration of or about 100 g / l. In a particularly preferred embodiment, the pharmaceutical composition will have a protein concentration of or about 102 g / l.

In another embodiment, the present invention provides a pharmaceutical composition of IgG comprising a protein concentration of between about 170 g / l and about 230 g / l. In certain embodiments, the protein concentration of the IgG composition is between about 180 g / l and about 220 g / l, preferably between about 190 g / l and about 210 g / l, most preferably about 200 g / l , or any suitable concentration within these ranges, for example approximately 170 g / l, 175 g / l, 180 g / l, 185 g / l, 190 g / l, 195 g / l, 200 g / l, 205 g / l, 210 g / l, 215 g / l, 220 g / l, 225 g / l, or 230 g / l. In a preferred embodiment, a pharmaceutical composition is provided having a protein concentration of or about 200 g / l.

The procedures provided herein allow the preparation of pharmaceutical compositions of IgG having very high levels of purity. For example, in one embodiment, at least about 95% of the total protein in a composition provided herein will be IgG. In other embodiments, at least about 96% of the protein is IgG, or at least about 97%, 98%, 99%, 99.5%, or more of the total protein in the composition will be IgG. In a preferred embodiment, at least 97% of the total protein in the composition will be IgG. In another preferred embodiment, at least 98% of the total protein of the composition will be IgG. In another preferred embodiment, at least 99% of the total protein of the composition will be IgG.

Similarly, the procedures provided herein allow the preparation of pharmaceutical IgG compositions containing extremely low levels of pollutants. For example, in certain embodiments, IgG compositions containing less than about 100 mg / l IgA are provided. In other embodiments, the IgG composition will contain less than about 50 mg / l IgA, preferably less than about 35 mg / l IgA, most preferably less than about 20 mg / l IgA.

 25

Typically, the pharmaceutical compositions provided herein will comprise one or more buffering agents or pH stabilizing agents suitable for intravenous, subcutaneous and / or intramuscular administration. Non-limiting examples of suitable buffering agents to formulate an IgG composition provided herein include glycine, citrate, phosphate, acetate, glutamate, tartrate, benzoate, lactate, histidine or other amino acids, gluconate, malate, succinate, formate, propionate. , carbonate, or any combination thereof adjusted to an appropriate pH. In general, the buffering agent will be sufficient to maintain an adequate pH in the formulation for a prolonged period of time. In a preferred embodiment, the buffering agent is glycine.

In some embodiments, the concentration of the buffering agent in the formulation will be between about 100 mM and about 400 mM, preferably between about 150 mM and about 350 mM, more preferably between about 200 mM and about 300 mM, most preferably approximately 250 mM. In a particularly preferred embodiment, the IVIG composition will comprise glycine between about 200 mM and about 300 mM, most preferably about 250 mM glycine. 40

In certain embodiments, the pH of the formulation will be between about 4.1 and about 5.6, preferably between about 4.4 and about 5.3, most preferably between about 4.6 and about 5.1. In particular embodiments, the pH of the formulation may be approximately 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 , 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or 5.6. In a preferred embodiment, the pH of the formulation will be between about 4.6 and about 5.1.

In some embodiments, the pharmaceutical compositions provided herein may optionally further comprise an agent for adjusting the osmolarity of the composition. Non-limiting examples of osmolarity agents include mannitol, sorbitol, glycerol, sucrose, glucose, dextrose, levulose, 50 fructose, lactose, polyethylene glycols, phosphates, sodium chloride, potassium chloride, calcium chloride, gluconoglucoheptonate calcium, dimethyl sulfone, and the like

Typically, the formulations provided herein will have osmolarities that are comparable to physiological osmolarity, approximately 285 to 295 mOsmol / kg (Lacy et al., Drug Information 55 Handbook - Lexi-Comp 1999: 1254. In certain embodiments, the osmolarity of the formulation will be between about 200mOsmol / kg and about 350mOsmol / kg, preferably between about 240 and about 300mOsmol / kg In particular embodiments, the osmolarity of the formulation will be about 200mOsmol / kg, or 210mOsmol / kg, 220mOsmol / kg, 230mOsmol / kg, 240mOsmol / kg, 245mOsmol / kg, 250mOsmol / kg, 255mOsmol / kg, 260mOsmol / kg, 265mOsmol / kg, 270mOsmol / kg, 275mOsmol / kg, 60 280mOsmol / kg, 285mOsmol / kg, 285mOmol / kg, 295mOsmol / kg, 300mOsmol / kg, 310mOsmol / kg, 320mOsmol / kg, 330mOsmol / kg, 340mOsmol / kg, 340mOsmol / kg, or 350mOsmol / kg.

The IgG formulations provided herein are, in general, stable in liquid form for a prolonged period of time. In certain embodiments, the formulations are stable for at least about 3 months at room temperature, or at least about 4, 5, 6, 7, 8, 8, 9,

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at room temperature. In general, the formulation will also be stable for 6 or at least about 18 months under refrigerated conditions (typically between about 2 ° C and about 8 ° C), or for at least about 21, 24, 27, 30, 33, 36, 39, 42 or 45 months under refrigerated conditions.

 5

IV. Treatment procedures

As routinely practiced in modern medicine, sterile preparations of concentrated immunoglobulins (especially IgG) are used to treat medical conditions that fall into these three main classes: immune deficiencies, inflammatory and autoimmune diseases, and acute infections. These IgG preparations 10 may also be useful for treating multiple sclerosis (especially recurrent-remitting multiple sclerosis or RRMS), Alzheimer's disease and Parkinson's disease. The purified IgG preparation of the present invention is suitable for these purposes, as well as other clinically accepted uses of IgG preparations.

The FDA has approved the use of IVIG to treat various indications, including bone marrow allograft transplantation, chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura (ITP), childhood HIV, primary immunodeficiencies, Kawasaki disease, chronic inflammatory demyelinating polyneuropathy (CIDP), and kidney transplant with a high antibody receptor or with an incompatible AB0 donor. In certain embodiments, the IVIG compositions provided herein are useful for the treatment or medical care of these diseases and conditions. twenty

In addition, patients outside of those indicated for IVIG are commonly provided for the treatment and medical care of various indications, for example, chronic fatigue syndrome, Clostridium difficile colitis, dermatomyositis and polymyositis, Graves ophthalmopathy, Guillain syndrome- Barre, muscular dystrophy, inclusion body myositis, Lambert-Eaton syndrome, lupus erythematosus, multifocal motor neuropathy, multiple sclerosis 25 (MS), myasthenia gravis, neonatal alloimmune thrombocytopenia, parvovirus B19 infection, pemphigus, post-transfusion purpura, renal transplant rejection, spontaneous / natural abortion, rigid person syndrome, opsoclonia-myoclonus, severe septicemia and septic shock in adult critically ill patients, toxic epidermal necrolysis, chronic lymphocytic leukemia, multiple myeloma, X-linked agammaglobulinemia, and hypogammaglobulinemia. In certain embodiments, the IVIG compositions provided herein are useful for the treatment or medical care of these diseases and conditions.

Finally, the experimental use of IVIG for the treatment and medical care of diseases that include primary immunodeficiency, RRMS, Alzheimer's disease and Parkinson's disease has been proposed (U.S. Patent Application No. US 2009/0148463 , which is incorporated herein by reference in its entirety for all purposes). In certain embodiments, the IVIG compositions provided herein are useful for the treatment or medical care of primary immunodeficiency, RRMS, Alzheimer's disease or Parkinson's disease. In certain embodiments comprising a daily administration, an effective amount to be administered to the subject can be determined by a doctor with consideration of the individual differences in age, weight, severity of the disease, route of administration (e.g. , intravenous versus subcutaneous) and response to treatment. In certain embodiments, an immunoglobulin preparation of the present invention can be administered to a subject at about 5 mg / kilogram to about 2000 mg / kilogram each day. In additional embodiments, the immunoglobulin preparation can be administered in amounts of at least about 10 mg / kilogram, at least 15 mg / kilogram, at least 20 mg / kilogram, at least 25 mg / kilogram, at least 45 30 mg / kilogram, or at least 50 mg / kilogram. In further embodiments, the immunoglobulin preparation can be administered to a subject in doses of up to 100 mg / kilogram, up to about 150 mg / kilogram, up to about 200 mg / kilogram, up to about 250 mg / kilogram, up to about 300 mg / kilogram, up to approximately 400 mg / kilogram every day. In other embodiments, the doses of the immunoglobulin preparation may be higher or lower. In addition, all 50 immunoglobulin preparations can be administered in one or more doses per day. Physicians familiar with the diseases treated by IgG preparations can determine the appropriate dose for a patient according to criteria known in the art.

In accordance with the present invention, the time required to complete a treatment cycle can be determined by a physician and can vary from only one day to more than one month. In certain embodiments, a treatment cycle can be from 1 to 6 months.

An effective amount of an IVIG preparation is administered to the subject intravenously. The term "effective amount" refers to an amount of an IVIG preparation that results in an improvement or remedy of the disease or condition in the subject. An effective amount to be administered to the subject can be determined by a doctor with consideration of the individual differences in age, weight, disease or condition being treated, severity of the disease and response to treatment. In certain embodiments, an IVIG preparation can be administered to a subject at a dose of about 5 mg / kilogram to about 2000 mg / kilogram per administration. In certain embodiments, the dose may be 65 at least about 5 mg / kg, or at least about 10 mg / kg, or at least about 20 mg / kg,

30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 950 mg / kg, 1000 mg / kg, 1100 mg / kg, 1200 mg / kg, 1300 mg / kg, 1400 mg / kg, 1500 mg / kg, 1600 mg / kg, 1700 mg / kg, 1800 mg / kg, 1900 mg / kg or at least about 2000 mg / kg. 5

The dosage and frequency of IVIG treatment will depend, among other factors, on the disease or condition being treated and the severity of the disease or condition in the patient. In general, for primary immune dysfunction, a dose of between about 100 mg / kg and about 400 mg / kg of body weight will be administered approximately every 3 to 4 weeks. For neurological and autoimmune diseases, up to 2 g / kg of body weight is implemented for three to six months during a five-day cycle once a month. In general, this is supplemented with a maintenance treatment comprising the administration of between approximately 100 mg / kg and approximately 400 mg / kg of body weight approximately once every 3 to 4 weeks. In general, a patient will receive a dose or treatment approximately once every 14 to 35 days, or approximately every 21 to 28 days. The frequency of treatment will depend, among other factors, on the disease or condition being treated and the severity of the disease or condition in the patient.

In a preferred embodiment, there is provided a method of treating an immunodeficiency, autoimmune disease, or acute infection in a human being in need thereof, the method comprising administering a pharmaceutical IVIG composition of the present invention. In a related embodiment, the present invention provides IVIG compositions manufactured in accordance with a method provided herein for the treatment of an immunodeficiency, autoimmune disease, or acute infection in a human being in need thereof.

In certain embodiments, immunodeficiency, autoimmune disease, or acute infection is selected from bone marrow allogeneic transplantation, chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura (ITP), childhood HIV, primary immunodeficiencies, Kawasaki disease, chronic inflammatory demyelinating polyneuropathy ( CIDP), kidney transplant with a high antibody receptor or an incompatible AB0 donor, chronic fatigue syndrome, Clostridium difficile colitis, dermatomyositis and polymyositis, Graves ophthalmopathy, Guillain-Barre syndrome, muscular dystrophy, inclusion body myositis , Lambert-Eaton syndrome, lupus erythematosus, 30 multifocal motor neuropathy, multiple sclerosis (MS), myasthenia gravis, neonatal alloimmune thrombocytopenia, Parvovirus B19 infection, pemphigus, post-transfusion purpura, renal transplant rejection, spontaneous / natural abortion, rigid man syndrome, opsoclonia-mine clony, severe sepsis or septic shock in critically ill patients, toxic epidermal necrolysis, chronic lymphocytic leukemia, multiple myeloma, X-linked agammaglobulinemia, hypogammaglobulinemia, primary immunodeficiency, RRMS, Alzheimer's disease, and Parkinson's disease 35.

Examples

The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that could be changed or modified to provide essentially the same or similar results.

Example 1

 Four. Five

The present example demonstrates that significant amounts of fibrinogen, amidolytic activity, precalicrein activity and lipoproteins can be removed from a modified slurry of fraction II + III pulp extracted by treatment with Aerosil before filtration.

Combustion silica (Aerosil 380) is currently used to adsorb fibrinogen, amidolytic activity, precalicrein activity and lipoproteins. To investigate the effect of Aerosil in more detail, six suspensions of fraction II + III modified with varying amounts of Aerosil were treated before filtration. In summary, a solution buffer containing 5 mM sodium acetate / 5 mM sodium dihydrogen phosphate buffer pH 4.5 was used to resuspend the modified II + III paste, prepared as described herein, in a proportion of 15 grams of dissolution buffer per gram of II + III paste. After the paste was added, the suspension was stirred for one hour at 2 ° C to 8 ° C in a controlled pH environment (IPC pH limits: 4.9 to 5.3). It has been found that the pH of this suspension normally moves to a pH of about 5.1 and, therefore, no additional pH adjustment is necessary. After further extraction, for at least 120 minutes, Aerosil 380, at 0 to 100 mg per gram of paste II + III, was added to the containers and the suspensions were incubated for one hour. Diatomaceous earth was added before depth filtration with a Cuno 50SA filter. After filtration, the filters were washed with extraction buffer containing 8 g l-1 of citrate and 0.2% polysorbate 80 (pH 5.0) and the wash was added to the filtrate. Then, the combined filtrate and the wash solution were treated with Polysorbate 80 to further solubilize hydrophobic impurities, for example lipoproteins, and the IgG was precipitated with 25% ethanol (pH 7) between -8 ° C and -10 ° C. The resulting Ppt G precipitate was almost white and possessed a higher IgG purity. Next, the precipitate was dissolved in purified water in a proportion of 7 grams of water per 2 grams of 65 Ppt G precipitate.

IgG solutions were analyzed to determine IgG recovery and impurities after the Cuno filtration stage. Specifically, the levels of amidolytic activity (PL-1), PKA activity, and fibrinogen (Table 3) were measured. Notably, as seen in Table 3, extraction protocols using 40 to 60 mg of Aerosil 380 per g paste of II + III resulted in acceptable levels of IgG recovery with significant decreases in amidolytic and PKA activity, as well as a significant decrease in the level of fibrinogen in the filtrate. Compared to extractions performed without Aerosil treatment, the addition of 40 mg of Aerosil 380 per gram of II + III paste resulted in a nearly 90% reduction in PKA activity and fibrinogen content and a 60% reduction in amidolytic activity, while a similar IgG recovery was maintained (73%).

 10

Table 1

Effect of the amount of Aerosil 380 in the filtration stage with Cuno II + III (extraction with conditions of GAMMAGARD® LIQUID)

 Spray

 IgG IgG PL-1 PKA Fibrinogen

 mg g-1 paste of II + III

 g l-1 plasma Recovery mmol min-1 g -1 protein (Ppt G) IU g-1 protein (Ppt G) mg g-1 protein (Ppt G)

 0

 4.9 72% 1.3 2403 19.5

 twenty

 5.2 82% 0.7 974 8.2

 40

 4.8 73% 0.5 290 2.1

 60

 4.8 71% 0.3 90 0.1

 80

 4.2 63% below the detection limit 37 0.0

 100

 4.3 65% below the detection limit 16 0.0

Example 2 15

The present example demonstrates that significant amounts of fibrinogen can be removed from a pulp suspension of fraction II + III modified by treatment with Aerosil before filtration. One purpose of the present experiment was to find suitable conditions for effective fibrinogen withdrawal without suffering significant IgG losses. twenty

The modified II + III paste, prepared according to the procedure provided herein, was dissolved in 5 mM sodium acetate / 5 mM monobasic sodium phosphate buffer pH 4.5. The proportion of the solution was 15 kg of buffer per 1 kg of II + III paste. The amount of acetic acid added was chosen so that the pH after sixty minutes of stirring was 4.9. To completely homogenize the suspension, it was stirred for up to twenty hours at 2 to 8 ° C before separating into 6 portions of 50 ml each in 100 ml beakers, in which varying amounts of Aerosil 380 were already present, such as Table 2 is given. Next, the solutions of the II + III suspension were stirred for 80 minutes in the presence of Aerosil, before processing and analysis. After stirring, all samples were centrifuged with a Heraeus Cryofuge 8500i at 4600 rpm for 30 minutes at 4 ° C in 50 ml Falcon tubes. 30

In this experiment, IgG measurements were taken using the nephelometry test, which was chosen due to its more precise values, compared to the ELISA test, at the high concentrations found in the suspension solutions of II + III. To minimize irritation of nonspecific turbidity, samples were filtered through 0.45 m filters before testing. For IgM, IgA and fibrinogen, ELISA tests were preferred due to the lower concentrations of these impurities in the suspension. The results of the experiment are shown in Table 2 below.

To further characterize the effect of Aerosil treatment on fibrinogen withdrawal and loss of IgG as described in example 1, Aerosil concentrations between 0 mg and 40 mg 40 per gram of modified II + III paste were additionally titled . The results shown in Table 2 confirm the high capacity of Aerosil to reduce fibrinogen in this fraction. Notably, the use of 40 mg per gram of II + III paste results in a nearly 90% reduction in fibrinogen, while the recovery of IgG in the filter cake is only reduced by 10%.

 Four. Five

Table 2.

Results of the variation of the dissolution conditions after the extraction of II + III with 5 mM NaAc / 5 mM NaH2PO4 pH 4.5 at a dissolution rate of 1 kg of II + III plus 15 kg of buffer after centrifugation fifty

 Values in the supernatant

 Dissolution Conditions

       Protein (Biuret) IgM ELISA lgG nef. IgA ELISA Fibrinogen ELISA

 Terms

    Additional stirring time (min) pH Conductivity (mS · cm-1) (g · l-1) (g) (g · l-1 plasma) μg · ml-1 (g · l-1 plasma) mg · ml- 1 (g · l-1 plasma) μg · ml-1 (g · l-1 plasma) μg · ml-1 (g · l-1 plasma)

 White 80 min

 mg · g-1 II + III paste 80 5.0 1.5 13.76 0.69 8.5 476 0.29 7.19 4.44 993 0.61 400 0.25

 Aerosil 5

 80 5.0 1.5 13.40 0.67 8.3 457 0.28 7.30 4.51 983 0.61 353 0.22

 Aerosil 10

 80 5.0 1.5 13.44 0.67 8.3 464 0.29 6.97 4.30 1009 0.62 272 0.17

 Aerosil 15

 80 5.0 1.5 12.01 0.60 7.4 451 0.28 7.04 4.35 1005 0.62 230 0.14

 Aerosil 30

 80 5.0 1.5 12.49 0.62 7.7 468 0.29 6.77 4.18 944 0.58 95 0.06

 Aerosil 40

 80 5.0 1.5 12.21 0.61 7.5 449 0.28 6.71 4.14 899 0.56 41 0.03

Example 3

The present example demonstrates the appropriate conditions that allow highly efficient extraction of IgG from a paste of the modified fraction II + III, while limiting levels of harmful impurities. 5 Specifically, the parameters including the concentration of acetic acid used in the II + III solution buffer and the treatment with Aerosil of the solution extracted before filtration were examined.

The paste of II + III was extracted in 5 mM sodium acetate, 5 mM sodium dihydrogen phosphate and varying amounts of concentrated acetic acid, as shown in Table 1, for 180 minutes at between 2 ° C and 8 ° C, followed by 10 addition of Aerosil 380 as shown in table 1. After one hour of stirring, the suspension was clarified by filtration with Cuno 50A in the presence of diatomaceous earth. Post-washing of the filter was carried out with the same buffer as for extraction except for the different amount of acetic acid, as given in Table 1, using 40% of the volume of the suspension before filtration. Precipitation of precipitate G was performed in the presence of 25% ethyl alcohol; 8 g · l-1 sodium citrate, and 0.2% Tween 80 (pH 7) at -8 ° C and after 8 hours of retention time, centrifugation was performed with Heraeus Cryofuge 8500i in glasses of stainless steel precipitates at 4600 rpm for 30 minutes at -10 ° C. The precipitate was dissolved in a ratio of 1: 2 in purified water.

Table 3. 20

The influence of the amount of acetic acid on the pH adjustment of the extraction buffer and the amount of Aerosil for the rinse on the yield and purity of IgG in Ppt G and the loss of IgG in the filter cake

 Aerosil (mg g-1 II + III) 0 20 40 40

 dissolution buffer

 acetic acid (g l-1) 0.40 0.40 0.40 0.51

 pH 4.52 4.52 4.52 4.46

 extract II + III

 pH 5.00 5.00 5.00 4.94

 conductivity (mS · cm-1) 1,340 1,340 1,340 1,344

 pre / post wash buffer

 acetic acid (g · l-1) 0.12 0.12 0.12 0.12

 pH 5.02 5.02 5.02 5.02

 II + III filtering

 CAE albumin (%) 11.9 12.5 12.3 10.7

 CAE α / β-globulin 25.5 22.9 18.9 17.2

 CAE denatured protein 3.2 0.0 0.0 0.0

 CAE (% γ-globulin) 59.4 64.6 68.8 72.1

 pH 5.00 5.03 5.04 4.92

 filter cake, loss of IgG

 (g · l-1 plasma) 0.09 0.20 0.56 0.29

 Dissolved PptG

 CAE albumin (%) 0.3 0.5 0.3 0.3

 CAE α / β-globulin 16.2 14.3 12.5 13.0

 CAE denatured protein 0.0 0.0 0.0 0.0

 CAE (% γ-globulin) 83.5 85.2 87.2 86.7

 fibrinogen (mg · l-1 plasma) 129 32 <4 4

 pKA (IU · mg-1 protein) 126 60 9 10

 PL-1 (μmol · ml-1 · g-1) 0.9 0.6 0.5 0.6

 PptG supernatant

 pH 7.20 7.35 7.30 6.96

 PptG supernatant, IgG loss

 (g · l-1 plasma) 0.14 0.13 0.11 0.09

As can be seen in Table 1, the addition of Aerosil to the pulp suspension of II + III has a marked influence on the purity of γ-globulin in the fraction of Ppt G. Without Aerosil, the purity of γ-globulin it is only 83.5%, while the addition of 40 mg of Aerosil per gram of II + III paste to the suspension of II + III paste increases the purity of γ-globulin to 87.2%. As evidenced, treatment with Aerosil results in a significant reduction in fibrinogen, PKA activity and amidolytic. A disadvantage of the adsorption of impurities in Aerosil is that the loss of IgG in the filter cake increases with increasing amounts of Aerosil. However, as shown in Table 1, higher concentrations of acetic acid in the extraction buffer partially offset the effect of Aerosil on the loss of IgG in the filter cake, and also reduce the loss of IgG in the fraction of Ppt G of supernatant. As can be seen in Table 1, the increase in the amount of acetic acid in the solution buffer from 400 ml per l of pasta to 510 ml per l of pasta reduced the amount of the loss of IgG in the cake. filter almost 50%. Advantageously, a higher concentration of acetic acid does not affect the purity of γ-globulin in the Ppt G fraction (87.2% pure using 400 ml per l of paste versus 86.7% using 510 ml per l of pasta). In addition, the results show that the difference in the pH value caused by the different amount of acetic acid is negligible, due to the high capacity of the acetic acid buffer near its pka value of 4.75 (Merck). This suggests that for better precision in large-scale manufacturing, acetic acid should be added by weight. Therefore, the influence of Aerosil on purity is much greater than the influence of acetic acid on purity, in the range investigated, as shown by the γ-globulin content measured by CAE.

Example 4 20

The results found in Example 3 suggested that the amount of IgG lost in the filter cake is strongly dependent on the amount of acetic acid used for the pH adjustment of the extraction buffer at a given Aerosil concentration. To characterize this effect, the modified II + III paste was extracted in purified water for approximately 120 minutes to obtain a homogeneous suspension and divided into 4 parts. These 25 parts were adjusted to pH 3.8, 4.2, 4.6, and 5.0, respectively, with 1 M acetic acid followed by a second extraction time of another 120 minutes. After this, the Aerosil treatment was performed with 40 mg of Aerosil 380 per gram of II + III paste. After one hour of stirring, the suspension was cleared by filtration with Cuno 50SA in the presence of diatomaceous earth. Post-washing of the filter was carried out with 100 percent of the volume of the suspension prior to filtration with extraction buffer adjusted to pH as given above. The filtrate was treated with 8 g · 1 -30 of sodium citrate and 0.2% Tween 80, adjusted to pH 7.0, and the IgG was precipitated with 25% alcohol at -8 ° C. The PptG precipitate was recovered by centrifugation at 4600 rpm for 30 minutes at -10 ° C in a Heraeus Cryofuge 8500i using stainless steel beakers. Next, the precipitate was dissolved in purified water in a proportion of 7 grams of water per 2 grams of Ppt G precipitate. Then, the relevant fractions were tested for IgG recovery, PKA activity, fibrinogen content and amidolytic activity, to determine the pH dependence of the recovery (table 4).

Table 4

Withdrawal dependent on fibrinogen pH, PKA and amidolytic activity by extraction and rinsing with 40 treatment with Aerosil

 G precipitate

 pH of acetic acid extraction and rinsing

 Acetic acid concentration after pH adjustment (mM) Loss of IgG in the filter cake (g · l-1 plasma) Loss of IgG in PptG supernatant (g · l-1 · plasma) Σ of IgG loss ( g · l-1 · plasma) PKA (IU · mg-1) Fibrinogen (mg · l-1 plasma) PL-1 (μmol · ml-1 · g-1)

 3.8

 75 0.02 0.85 0.87 138 243 -

 4.2

 25 0.04 0.53 0.57 35 182 -

 4.6

 10 0.07 0.05 0.12 2 99 2

 5.0

 5 0.19 0.06 0.25 0.6 3 0.5

 3.8

 75 - - - 116 304

 3.8

 4.2

 25 0.02 0.34 0.36 44 313 2.6

 4.6

 10 0.04 0.52 0.56 8 148 1.7

 5.0

 5 0.14 0.09 0.23 3 10 <0.2

Table 4 shows that the withdrawal of PKA activity, amidolytic (PL-1), and fibrinogen with Aerosil is less effective at lower pH during rinsing. From the results obtained here, it can be seen that the most effective pH for the effective withdrawal of PKA, amidolytic (PL-1), and fibrinogen activity, while maintaining an efficient IgG recovery in the Ppt G fraction, is approximately pH 5. High concentrations of acetic acid give rise to

a significant IgG loss in the Ppt G supernatant (0.85 g l-1 in the presence of 75 mM acetic acid) while the loss of IgG in the filter cake is minimized.

Example 5

 5

To determine the dependence of the Aerosil treatment on the results found in Example 4, the experiment was repeated, but with the Aerosil treatment stage omitted. In summary, the modified II + III paste was extracted in purified water for approximately 120 minutes to obtain a homogeneous suspension and divided into 4 parts. These parts were adjusted to pH 3.8, 4.2, 4.6, and 5.0, respectively, with 1 M acetic acid followed by a second extraction time of another 120 minutes. After this, the suspension was cleared by filtration with Cuno 10 50SA in the presence of diatomaceous earth. Post-washing of the filter was carried out with 100 percent of the volume of the suspension prior to filtration with extraction buffer adjusted to pH as given above. The filtrate was treated with 8 g · l-1 sodium citrate and 0.2% Tween 80, adjusted to pH 7.0, and the IgG was precipitated with 25% alcohol at -8 ° C. The PptG precipitate was recovered by centrifugation at 4600 rpm for 30 minutes at -10 ° C in a Heraeus Cryofuge 8500i using stainless steel beakers. Next, the precipitate was dissolved in purified water in a proportion of 7 grams of water per 2 grams of Ppt G precipitate. Then, the relevant fractions were tested for IgG recovery, PKA activity, contained in fibrinogen and amidolytic activity, to determine the pH dependence of the recovery (table 5).

Table 5. 20

Withdrawal dependent on fibrinogen pH, PKA and amidolytic activity by extraction and rinsing without Aerosil treatment

 G precipitate

 pH of acetic acid extraction and rinsing

 Acetic acid concentration after pH adjustment (mM) loss of IgG in the filter cake (g l-1 plasma) loss of IgG in PptG supernatant (g l-1 plasma) Σ loss of IgG (g l-1 plasma) PKA (IU mg-1) Fibrinogen (mg l-1 plasma) PL-1 (μmol ml-1 g-1)

 3.8

 75 0.00 0.42 0.42 214 173 3.9

 4.2

 25 0.01 0.19 0.20 170 134 3.2

 4.6

 10 0.01 0.09 0.10 38 193 1.4

 5.0

 5 0.09 0.04 0.13 6.4 114 0.4

 3.8

 75 0.00 0.53 0.53 193 355

 3.8

 4.2

 25 0.00 0.28 0.28 171 346 3.2

 4.6

 10 0.01 0.14 0.15 131 340 1.5

 5.0

 5 0.16 0.05 0.21 5.3 189 <0.2

 25

Consistent with the results found in example 4, the increase in the pH of the extraction / dissolution buffer to 5.0 resulted in a small increase in the IgG lost in the filter cake, however, this loss was more than compensated by a greater decrease in the loss of IgG in the supernatant of Ppt G.

When the results of Example 4 and Example 5 are compared, it can be seen that the treatment with Aerosil 30 reduces the amount of residual PKA activity found in the fraction of dissolved PptG when the paste of II + III is extracted at lower pH ( 3.8, 4.2 and 4.6), but not at pH 5.0 (Figure 2). On the contrary, the treatment with Aerosil significantly reduces the fibrinogen content of the dissolved Ppt G fraction when the paste of II + III is extracted at higher pH (Figure 3; compare pH 4.6 and 5.0 with pH 3, 8 and 4.2). Aerosil treatment does not appear to affect the level of residual amidolytic activity found in the dissolved Ppt G fraction (Figure 4). Notably, the level of the three pollutants in the dissolved Ppt G fraction is greatly reduced when the paste of II + III is extracted at pH 5.0, compared to pH 3.8, 4.2 and 4.6.

Example 6

 40

As can be seen in the previous examples, the losses of IgG in the filter cake and supernatants of Ppt G are minimized when the paste of II + III is extracted at a pH of about 4.5 to 4.6. However, it is also evident in the previous examples, that the critical impurities, including PKA activity, amidolytic activity, and fibrinogen content, are much higher when the paste of II + III is extracted at pH 4.5 or 4, 6 compared to when the extraction occurs at a pH of about 4.9 to 5.0. Accordingly, the present example 45 was performed to determine if the higher levels of impurities observed when pulping II + III at pH 4.5 could be compensated by increasing the amount of Aerosil used to adsorb contaminants, while maintaining levels low loss of IgG in the filter cake and supernatant of Ppt G.

In this sense, the modified II + III paste was extracted as before, with the extraction buffer having a pH of 4.5. Next, increasing amounts of Aerosil 380 were added, up to 200 mg per g paste of II + III, and the suspension was stirred for one hour. An additional sample processing was performed as before.

As can be seen in Table 6, the removal of both fibrinogen and PKA activity is significantly improved by rinsing with high amounts of Aerosil. The losses of IgG in the filter cake due to the binding on Aerosil are increased with high amounts of Aerosil, although this effect was somewhat compensated by the decrease in the loss of IgG in the supernatant of Ppt G. However, of Significantly, the amidolytic activity could not be reduced by the high amounts of Aerosil when the paste of II + III was extracted at pH 4.5. In addition, although the purity of γ-globulin is improved with higher amounts of Aerosil, in all cases it is still below the specification limit of> 86% for Ppt G, probably due to the low pH at extraction and cleared up.

Table 6.

 fifteen

Table 6 gives the results of the variation in the concentration of Aerosil with extraction and rinse of pH 4.5

 G precipitate

 Amount of Aerosil in mg per gram II + III

 loss of IgG in the filter cake (g l-1 plasma) loss of IgG in PptG supernatant (g l-1 plasma) Σ loss of IgG (g l-1 plasma) PKA (IU mg-1) Fibrinogen (mg l-1 plasma) PL-1 (μmol ml-1 g-1) CAE (% gamma globulin)

 0

 0.01 0.21 0.22 177.1 272 5.4 69.1

 40

 0.13 0.50 0.63 9.3 173 1.8 76.8

 100

 0.11 0.30 0.41 0.1 76 6.1 79.4

 200

 0.25 0.08 0.33 below the limit of det. 10 4.5 84.1

Example 7

 twenty

The present example demonstrates the effect of the pH of the extraction buffer on the removal of impurities after resuspension and rinsing of the paste of II + III.

Low pH extraction of the modified II + III paste at pH 4.2 was performed using a ratio of 15 grams of buffer per gram of II + III paste. Next, the suspension was divided into 3 parts and the pH was adjusted to 4.5, 4.7, or 5.0 respectively with 3M Tris. After this, each solution was further divided into two parts, which were incubated for one hour at 4 ° C or at 25 ° C. Filtration with Cuno 50 (90) SA was performed using 10 mM sodium acetate post-wash buffer having the same pH as the respective rinse buffer. The filtrates were treated with 8 g l-1 citrate and 0.2% Tween 80, and then the IgG was precipitated by the addition of 25% ethyl alcohol at -10 ° C for at least 8 hours. The precipitate was recovered by centrifugation as previously described, 30 and the precipitate was dissolved in one volume in duplicate of purified water. The resulting suspension was filtered using a Cuno VR06 filtration device, in a final solution having a conductivity of approximately 1.3 mS cm -1.

To assess the various conditions, the levels of IgG, IgA, IgM, transferrin, fibrinogen and other impurities were determined. The results of the analysis are given in Table 7, which shows the pH dependence of the rinse pH of the II + III suspension. Within the pH range of 4.5 to 5.0 the amount of IgA, IgM, transferrin and fibrinogen does not vary over a wide range, but other unwanted proteins are present at higher levels when buffers with lower pH are used. It was calculated that these other impurities comprise 10% of the total protein at pH 4.5, but less than 1% at pH 5. The IgG content is the highest at pH 5.0, while dependence 40 on the IgG content temperature and impurity levels, between 4 ° C and 25 ° C, is negligible.

Table 7.

Table 7 compares the reduction of impurities of dissolved II + III with the filtering of VR06 by the variation in pH and temperature during 1 hour of incubation after resuspension of the paste of II + III and subsequent filtration after low pH extraction without additives. 5

 Incubation pH and both stages of filtration

 incubation temperature and first stage of filtration stage% protein

 IgG by nephelometry

 IgA by ELISA IgM by ELISA transferrin by ELISA fibrinogen by ELISA other impurities calculated

 4,5

 4 ºC II-III dissolved 55.0 7.9 3.6 1.3 3.0 29.3

 filtered with Cuno 90

 51.7 8.5 4.0 1.6 2.5 31.7

 Ppt G dissolved

 64.4 8.3 4.4 0.1 2.8 20.0

 VR06 filtering

 71.8 9.5 5.1 0.1 3.0 10.6

 25 ° C

 II + III dissolved 55.0 7.9 3.6 1.3 3.0 29.3

 filtered with Cuno 90

 52.3 8.0 4.2 1.6 2.6 31.3

 Ppt G dissolved

 64.5 7.4 4.8 0.1 3.8 19.4

 VR06 filtering

 72.5 9.1 5.0 0.1 3.3 9.9

 4.7

 4 ºC II + III dissolved 55.0 7.9 3.6 1.3 3.0 29.3

 filtered with Cuno 50

 49.4 8.2 3.7 1.6 2.5 34.6

 Ppt G dissolved

 68.8 9.5 5.0 0.1 3.5 13.2

 VR06 filtering

 76.5 9.8 5.4 0.1 3.5 4.7

 25 ° C

 II + III dissolved 55.0 7.9 3.6 1.3 3.0 29.3

 filtered with Cuno 50

 49.6 8.6 3.7 1.5 3.1 33.6

 Ppt G dissolved

 66.9 8.8 4.9 0.1 4.1 15.2

 VR06 filtering

 75.8 10.2 5.3 0.1 4.3 4.3

 5.0

 4 ºC II + III dissolved 55.0 7.9 3.6 1.3 3.0 29.3

 filtered with Cuno 50

 52.5 9.3 3.4 1.7 2.1 31.0

 Ppt G dissolved

 76.9 10.1 4.9 0.1 3.0 5.0

 VR06 filtering

 83.6 10.6 4.5 0.1 3.0 0.0

 25 ° C

 II + III dissolved 55.0 7.9 3.6 1.3 3.0 29.3

 filtered with Cuno 50

 53.4 8.9 3.4 1.8 2.6 30.0

 Ppt G dissolved

 76.6 10.3 4.8 0.1 3.9 4.3

 VR06 filtering

 79.9 10.9 4.7 0.1 3.6 0.8

As can be seen in Table 8, additional analyzes of the fraction of dissolved Ppt G and filtered VR06 indicate that the use of buffers having a pH of 4.5 for the rinse stages of II + III and filtration treatment of II + III, results in an increase in the level of aggregates and low molecular weight components. This effect is further enhanced when the steps are carried out at 25 ° C, instead of at 4 ° C. On the contrary, when the samples are treated at higher pH (pH 5.0), the resulting Ppt G suspensions and filtrate contain material levels of ~ 350 kDa (IgG or dimeric IgA) higher than those of the solutions treated at pH 4.5 (table 8). In addition, treatments with lower pH resulted in higher levels of amidolytic activity than with the pH 5.0 treatment.

 fifteen

Table 8

Influence of pH and temperature during the incubation and filtration of the suspension of the paste of II + III on the activities of PKA and amidolytic of the precipitate G dissolved and on the distribution of molecular size in the filtrate VR06. twenty

 Incubation pH and both stages of filtration

 incubation temperature and first filtration stage dissolved Ppt G filtered VR06

 PKA activity

 amidolytic activity (PL-1) molecular size distribution (%)

 pH

 temp. ºC IU ml-1 IU g-1 nmol · ml-1 · min-1 nmol · min-1 · g-1> 450 kDa ~ 350 kDa ~ 160 kDa <60 kDa

 4,5

 4 60.2 2028 171.8 5788 28.33 6.61 63.21 1.85

 4,5

 25 103.4 3569 239.4 8264 29.44 6.02 61.76 2.79

 4.7

 4 1343.2 38029 152.1 4306 23.71 8.82 65.82 1.64

 4.7

 25 2215.9 67702 180.2 5506 24.54 8.02 65.06 2.38

 5.0

 4 134.5 4009 67.6 2015 17.81 10.78 70.03 1.38

 5.0

 25 186.6 5738 87.3 2685 19.16 10.54 68.84 1.46

Example 8

The results presented in Tables 7 and 8 show that resuspension of the II + III precipitate should be performed at refrigerated temperatures (2 ° C to 8 ° C) and that the pH should be maintained at pH 5.0 to minimize the dissolution of the 5 components of high (> 450 kDa) and low (> 70 kDa) molecular weight, as well as components that have amidolytic activity. Effective rinsing after the suspension of the II + III paste will reduce the impurity load for subsequent processing by Ppt G chromatography and, therefore, is key to meeting the specifications of the final IVIG container reproducibly. To further validate this finding, the modified II + III paste was dissolved and processed, as before, the modified II + III paste paste was dissolved at pH 4.2 and then adjusted to pH 4.5, 4.7 or 5.0. As seen in Table 9, the IgM is removed more efficiently by a pH of 5.0 during suspension and rinsing of the II + III paste than the removal at pH 4.5. In this experiment, the IgG yield is similar to pH 5.0 and 4.5.

Table 9 15

Performance of IgG, IgA, IgM, transferrin and fibrinogen in various stages of resuspension of the paste of II + III, filtration and precipitation of Ppt G

 % performance

 Incubation pH and both stages of filtration

 incubation temperature and first stage of filtration stage Total IgG protein by nef./ ELISA1 IgA by ELlSA IgM by ELISA transferrin by ELISA fibrinogen by ELISA

 4,5

 4 ºC II + III dissolved 100.0 100.0 100.0 100.0 100.0 100.0

 filtered with Cuno 50

 100.2 92.9 100.2 97.8 118.7 86.3

 filtered with Cuno 90

 97.0 91.1 105.3 108.6 123.2 80.1

 Ppt G supernatant

 33.5 4.4 - - - -

 Ppt G dissolved

 71.7 83.9 75.6 89.6 5.0 66.5

 VR06 filtering

 65.3 85.2 78.8 93.1 5.2 64.8

 25 ° C

 II + III dissolved 100.0 100.0 100.0 100.0 100.0 100.0

 filtered with Cuno 50

 101.0 92.3 100.9 104.4 113.2 96.7

 filtered with Cuno 90

 97.2 92.4 98.9 113.4 123.9 84.6

 Ppt G supernatant

 33.8 5.1 - - - -

 Ppt G dissolved

 72.9 85.5 68.8 97.5 4.7 89.9

 VR06 filtering

 68.3 90.0 78.9 95.9 5.5 75.0

 4.7

 4 ºC II + III dissolved 100.0 100.0 100.0 100.0 100.0 100.0

 filtered with Cuno 50

 93.5 83.9 98.0 98.0 115.9 75.3

 Ppt G supernatant

 27.5 2.7 - - - -

 Ppt G dissolved

 64.8 81.1 78.2 90.2 4.5 73.9

 VR06 filtering

 60.5 84.1 75.0 92.1 5.1 70.2

 twenty

(Continuation)

 % performance

 Incubation pH and both stages of filtration

 incubation temperature and first stage of filtration stage Total IgG protein by nef./ ELISA1 IgA by ELlSA IgM by ELISA transferrin by ELISA fibrinogen by ELISA

 4.7

 25 ºC II + III dissolved 100.0 100.0 100.0 100.0 100.0 100.0

 filtered with Cuno 50

 94.7 85.3 103.1 98.5 113.8 95.8

 Ppt G supernatant

 28.5 4.1 - - - -

 Ppt G dissolved

 65.3 79.5 73.1 90.9 5.0 87.0

 VR06 filtering

 61.9 85.3 80.7 93.0 5.3 86.9

 5.0

 4 ºC II + III dissolved 100.0 100.0 100.0 100.0 100.0 100.0

 filtered with Cuno 50

 86.1 82.3 101.5 81.5 117.5 59.8

 Ppt G supernatant

 26.0 4.1 - - - -

 Ppt G dissolved

 58.1 81.2 74.8 79.3 4.5 57.0

 VR06 filtering

 54.0 82.1 72.5 67.8 4.7 52.5

 25 ° C

 II + III dissolved 100.0 100.0 100.0 100.0 100.0 100.0

 filtered with Cuno 50

 88.0 85.4 99.7 83.2 124.1 75.4

 Ppt G supernatant

 23.4 1.5 - - - -

 Ppt G dissolved

 60.5 84.2 79.0 81.6 4.5 78.2

 VR06 filtering

 59.0 85.7 82.0 77.6 4.5 69.0

Example 9

 5

To evaluate the optimum pH at the stage of extraction of the paste of II + III, for minimized proteolytic activities in the filtrate, the pH was varied during extraction and filtration over a wider range of pH 3.8 to 7.8. For this purpose, the modified II + III paste was extracted at low pH in a ratio of 1 + 8. After a short stirring time, to obtain a homogeneous dispersion, the suspension was divided into 8 parts, the pH was adjusted with acetic acid or Tris buffer to pH 3.8, 4.2, 4.6, 5.0, 6.6, 7.0, 7.4 or 7.8, and extracted for an additional 120 minutes. 10 After this, the pH was adjusted to 5.1 and the rinse was performed by centrifugation in 50 ml Falcon tubes. Precipitation of Ppt G was performed under standard conditions. Amidolytic and PKA activity in the dissolved fraction of Ppt G was measured as indicated in Figures 5 and 6.

As can be seen in the sample stored at 4 ° C in Figure 5, amidolytic activity is minimized when the paste of II + III is extracted at pH 5.0. Stressing also the issue that the samples should not be kept at elevated temperature (room temperature) for prolonged periods of time, the amidolytic activity was elevated after storage of the dissolved fraction of Ppt G at room temperature for one week. Similarly, as seen in Figure 6, PKA activity is minimized when the paste of II + III is extracted at pH 5.0 or more. twenty

Example 10

The present example evaluates the pH dependence on the loss of IgG yield during extraction and rinsing. In summary, 110 grams of modified II + III paste were resuspended in a proportion of 15 grams of purified water per gram of II + III paste, followed by extraction for 120 minutes. Then, the sample was divided into four parts and the pH was adjusted with acetic acid to pH 3.8, 4.2, 4.6 or 5.0. Pre-extraction was performed before dividing the original pH into four parts to guarantee four identical parts, which were then adjusted to the mentioned pH and were also extracted for an additional hour. The samples were then rinsed by filtration with Cuno 50SA at the same pH used for each extraction and precipitation of Ppt G. After filtration with Cuno, all parts were treated in the same way, which means precipitation of the standard G precipitate a 30 pH 7.0 for all parts. The results of two of these experiments are summarized in Tables 10 and 11 below.

Table 10

Recovery of protein and IgG in the filtrates as well as the results of MSD and CAE of the redissolved G precipitates

 5

 38/1 41

 Extraction and rinse pH 3.8 4.2 4.6 5.0 3.8 4.2 4.6 5.0

 Acetic acid concentration after pH adjustment 75 25 10 5 75 25 10 5

 Filtrate protein recovery (%) 100.3 99.6 92.8 76.0 106.4 107.5 99.7 81.0

 IgG recovery (%) 87.6 89.8 90.3 86.0 96.2 97.3 95.1 88.3

 G precipitate

 MSD (% area)> 450 kDa 19.2 20.5 14.8 13.2 22.5 22.8 20.7 16.8

 ~ 350 kDa

 8.7 7.6 10.5 11.7 5.2 5.0 7.7 9.4

 ~ 160 kDa

 69.5 69.2 72.9 74.2 66.0 65.6 67.5 72.0

 <70 kDa

 2.7 2.7 1.8 1.0 6.2 5.6 4.1 1.9

 CAE (% gammaglobulin)

 67.4 68.3 78.1 86.7 68.3 69.8 77.5 85.1

Table 11

Loss of IgG during extraction and rinsing as well as proteolytic and fibrinogen activities in the redissolved G precipitate 10

 G precipitate

 pH of acetic acid extraction and rinsing

 acetic acid concentration after pH adjustment (mM) loss of IgG in the filter cake (g · l-1 · plasma) loss of IgG in plasma PptG supernatant Σ loss of plasma IgG) PKA (lU mg- 1) fibrinogen (mg · l-1 · plasma) PL-1 (μmol ml-1 g-1)

 3.8

 75 0.00 0.42 0.42 214 173 3.9

 4.2

 25 0.01 0.19 0.20 170 134 3.2

 4.6

 10 0.01 0.09 0.10 38 193 1.4

 5.0

 5 0.09 0.04 0.13 6.4 114 0.4

 3.8

 75 0.00 0.53 0.53 193 355

 3.8

 4.2

 25 0.00 0.28 0.28 171 346 3.2

 4.6

 10 0.01 0.14 0.15 131 340 1.5

 5.0

 5 0.16 0.05 0.21 5.3 189 0.2

The above data confirm the results shown in Figures 5 and 6 on the activation of proteolytic enzymes by extraction at low pH. Taken together, the data demonstrate that extraction at low pH causes less loss of IgG due to the increase in the solubility of all proteins. These results are consistent with the examples provided above. Additionally, the IgG losses shown in Table 11 suggest that higher concentrations of acetic acid, which result in lower pH, result in less loss of IgG in the filter cake but greater loss of IgG in the precipitate supernatant G. This phenomenon can be explained by the higher concentrations of acetate in the precipitation stage after extraction at low pH. twenty

Example 11

To determine the appropriate II + III extraction conditions, a protocol was compared using low pH extraction with purified water adjusted with acetic acid at pH 4.3 and readjustment to pH 4.9 before filtration with the extraction in 5 mM sodium acetate / 5 mM sodium dihydrogen phosphate with 600 g of acetic acid per 1000 liters of extraction buffer, resulting in a solution pH of 4.8 to 4.9. The experiments were performed on a pilot scale, starting with 3.8 to 5 kg of the modified II + III paste. All experiments included treatment with Aerosil with 40 g of Aerosil per kg of II + III paste. For rinsing, a Strassburger filter press with 30 cm x 30 cm filter racks equipped with Cuno 50SA filter sheets was used. Post-wash was performed with 4 dead volumes of the filter-press, with a 5 mM sodium acetate / dihydrogen phosphate buffer

of 5 mM sodium with 150 grams of acetic acid per 1000 liters of buffer. Centrifugation of precipitate G was performed with a Cepa® Z61H centrifuge at 17000 rpm (rotor diameter of 10.5 cm) at a flow rate of 40 liters per hour. The results of the experiment, performed in triplicate, are shown in Table 12.

Table 12 5

Comparison of extraction at low pH at pH 4.3 and change from pH to 4.9 prior to treatment with Aerosil with extraction by GAMMAGARD® LIQUID conditions improved with 600 g of glacial acetic acid per 1000 l of extraction buffer

 10

 experiment

 unit P00809NG2 F00909NG2 P01509NG2 P02209NG2 P02509NG2 P02609NG2

 process

    extraction at pH under 600 g HAc per 1000 l extraction buffer

 II + III paste

 Lot No. VNEGJ039 VNGBJ022 VNGBJ216 VNGBJ216 VNGBJ022 VNEGJ039

 kg 5.00 5.00 4.97 4.96 4.96 3.86

 PEQ

 l 106.65 118.70 123.32 123.31 117.65 82.35

 extract II + III

 protein

 g 1316 1310 1364 1336 1347 1022

 protein yield (BIURET)

 g · l-1 12.3 11.0 11.1 10.8 11.5 12.4

 IgG yield (ELISA)

 g · l-1 6.09 5.89 6.25 5.50 6.06 6.38

 IgG yield (ELISA)

 g · l-1 1.06 0.96 0.98 1.00 1.01 1.10

 IgG yield (ELISA)

 g · l-1 0.40 0.40 -0.47 0.40 0.43 * 0.48

 filtered out

 protein

 g 957 1007 1038 955 926 731

 protein yield (BIURET)

 g · l-1 9.0 8.5 8.4 7.7 11.5 12.4

 IgG yield (ELISA)

 g · l-1 5.7 5.62 5.45 5.02 6.06 6.38

 IgG yield (ELISA)

 g · l-1 1.03 0.88 0.96 0.90 1.01 1.10

 IgG yield (ELISA)

 gL-1 0.32 0.39 037 0.27 0.43 0.48

 filter cake extract

 IgG yield (ELISA)

 g1-1 - - - 0.07 0.07 0.07

 Ppt G supernatant

 protein

 g 196 130 188 197 168 109

 protein yield (BIURET)

 g · l-1 1.8 1.1 1.5 1.5 1.4 1.3

 IgG yield (ELISA)

 gL-1 0.05 0.02 0.03 0.05 0.03 0.04

As seen in Table 12, both extraction protocols result in similar IgG recoveries. Given the results of examples 3 to 10, which show that various impurities can be minimized by extracting the paste of II + III at pH 5.0, the results provided in Table 12 show that the extraction at pH of 4, 8 to 4.9 with 600 g of glacial acetic acid per 1000 l of extraction buffer is superior to the extraction at pH below 15 and the subsequent adjustment to pH of 4.8 to 5.0.

Example 12

During manufacturing, the racks and lines of the filter press that are connected to and from the tanks have a significant volume of vacuum, which is still filled with suspension or filtering before the post-wash begins. When the post-wash is finished, this vacuum volume remains in the filter press. The standard protocols represent this volume of vacuum washing with between one and two dead volumes of the filter-press. To determine whether standard postfilter washes allow for effective recovery of total IgG, experiments were performed using varying volumes of the wash buffer in large-scale manufacturing IgG purifications. Figure 1 shows the dependence of postwash volumes (measured by dead or vacuum volumes) on the levels of residual IgG and total protein.

Notably, as seen in Figure 1, a post-filtration wash using approximately a dead volume of filter-press in duplicate results in a significant loss of IgG recovery, since the wash solution contains approximately 1.5 grams of IgG per l of wash solution. Surprisingly, it was discovered that a 3.6-fold filter-press dead volume of post-wash buffer is required for a

efficient recovery of IgG (indicated by an arrow in figure 1). Additional post-washing of more than 3.6 dead volumes of the filter press is not convenient, as it will result in dilution of the filtrate without the recovery of additional IgG.

Example 13 5

During the precipitation stage of II + III, the alcohol concentration was increased from 20 to 25% and the temperature was reduced from -5 ° C to -7 ° C. When the II + III paste was dissolved, at least 600 ml of glacial acetic acid per 1000 l of volume was used to adjust the pH of the II + III paste resuspension buffer, unlike the previous use of the proportion 510 ml of glacial acetic acid / 1000 l buffer. The extraction rate was re 1 + 15 with the 10 acetic acid buffer. For rinsing, 0.04 to 0.06 grams of Aerosil (typically, at the lower end of this range, for example, 0.04 g) was added for each gram of the paste of II + III. For post-wash, four (4 x) dead volumes of post-buffer wash filter-press were used. For example, 4.3 x dead volumes of filter-press were used in a particular experiment while 3.6 x volumes were used in another experiment. Dead volumes four times or more increased from dead volumes 1.8 x previously used. The buffer was adjusted with 150 ml of glacial acetic acid per 1000 l of buffer, an increase over 120 ml of glacial acetic acid / 1000 l of previously used buffer. These changes resulted in an 8% higher IgG yield and a purity of at least 86% γ-globulin. A very low residual amount of IgG was discovered in the filter cake extract, when extracted with 0.1 M sodium phosphate + 150 mM NaCl (pH 7.4, conductivity 25.5 mS / cm). twenty

Example 14

A. Optimization of fractionation I: addition of ethanol by spraying versus addition as a flowing agent; pH adjustment to pH 7.0 or 7.5 after the addition of ethanol 25

Table 13 shows the performance of IgG by the manufacturing processes currently in use and provides a comparison reference in the experiments described below. 15-20% IgG is lost from the Cohn pool to the filtrate. Approximately 0.4 g of IgG are lost per liter of plasma in the II + III supernatant.

 30

Table 13

 IgG Performance (2009)

    g / l plasma% Cohn reserve

 Fraction

 LA B1 (source) Vienna (source) LA B5 (Rec.) L.A. (source) Vienna (source) L.A. (Rec.)

 Cohn Reserve

 6.18 (5.28-7.02) 6.26 (5.43-6.68) 7.49 * (6.41-8.41) 100 100 100

 supernatant II + III

 0.41 (0.23-0.63) 0.39 (0.33-0.47) 0.37 (0.23-0.48) 6.6 6.2 4.9

 precipitate II + III (calculated)

 5.77 5.87 7.12 93.4 93.8 95.1

 filtered II + III

 4.93 (4.21-5.57) 5.19 (4.11-5.48) 6.43 (6.11-7.02) 80 83 86

 * LA B5, Cohn reserve of recovered plasma: lower concentration of IgG due to saline solution. Average of LA B1, Cohn reserve of recovered plasma: 8.52 g / l.

Process

 35

The Cohn pool was thawed at 24-27 ° C for 6-7 hours in a 14-liter bucket. After this, the material was mixed overnight at 2-8 ° C. Next, the reserve was divided into four parts (800 g each):

1: addition of ethanol as a flowing agent, followed by pH adjustment to 7.0

2: addition of ethanol as a flowing agent, followed by pH adjustment to 7.5

3. Addition of ethanol by spraying, followed by pH adjustment to 7.0 40

4. addition of ethanol by spraying, followed by pH adjustment to 7.5

First, all parts were cooled to 0 ° C. Then, 8% ethanol was added to parts 1 and 2 as a flow and by spraying to parts 3 and 4 using a spray head. In both procedures ethanol was added at approximately the same rate. During the addition of ethanol, the cryostat was adjusted to -5 ° C and 75 ml of ethanol was added to each part while mixing. The pH was adjusted to 7.0 or 7.5 by 1 M acetic acid. The solution was then incubated for 1 hour. After incubation, the solution was centrifuged with a centrifuge with beaker (4600 rpm; 30 min; -2 ° C).

Results

The IgG yields were measured nephelometrically and are shown in Table 14. Almost 100% of the IgG yield was obtained in the fractionation supernatant I with the improved procedure (ethanol spraying) while with the conventional addition of ethanol. 0.2 to 0.25 g / l plasma was lost. These results indicate that the improved process can lead to an increase in IgG yield of up to 0.2 g / l plasma in manufacturing.

Table 14

 Sample

 Weight (g) IgG per nef. (average of 3)

 mg / ml g% Purity (%) g / l plasma

 Plasma devoid of cryoprecipitate

 800 5.72 4.57 100.00 12.2 5.72

 Supernatant 1

 856 5.10 4.37 95.57 12.6 5.46

 Supernatant 2

 853 5.16 4.41 96.36 12.7 5.51

 Supernatant 3

 853 5.36 4.58 100.14 13.1 5.72

 Supernatant 4

 848 5.33 4.52 98.90 13.0 5.65

 10

B. Pilot scale: fractionation I (spray versus flow; pH adjustment to 7.4 after the addition of ethanol) and fractionation II + III (pH adjustment to 6.7 before the addition of ethanol, readjustment to 6 , 9 after the addition of ethanol)

Example 15

 fifteen

Process

2.8 kg of plasma was thawed while mixing at 2 ° C. Fraction I: 8% ethanol was added and the pH was adjusted to 7.4 using 5M acetic acid. While mixing, the suspension was cooled to a temperature of -2 ° C. Spray conditions were obtained using a spray head. In both procedures, the addition of ethanol and 5 M acetic acid was performed at approximately the same rate. After 1 hour incubation, the solution was centrifuged using a CEPA centrifuge at a temperature of -4 ° C.

Fraction II + III: the pH was adjusted to 6.7 using a buffer of pH 4, then 25% ethanol (1) was added by spraying or (2) as a flow as was done conventionally. Then, the pH was readjusted to 6.9. The incubation was carried out for 10 hours at -7 ° C.

Results

The loss of IgG during fraction II + III was measured nephelometrically with 25% ethanol and is shown in Table 15. 30 The IgG measurements had a certain variation, so the average value of the optimized procedure was taken.

Table 15

 Fraction I, loss of IgG Supernatant of fraction II + III, ethanol added to 25% IgG in the filtrate

 Experiment

 g per liter of plasma g per liter of plasma% Cohn reserve

 NG2C73

 0.47 0.08 85.28

 NG2C73-1

 0.34 0.03 92.27

 NG2C73-2

 0.05 0.06 95.94

 Average

 0.29 0.06 91.16

 Reference (addition of ethanol as a flowing agent)

 0.29 0.10 87.38

Up to the point of the precipitate of fraction II + III, only 0.35 g IgG / l plasma was lost. An increase in yield of 0.04 g IgG per liter of plasma was achieved during fractionation II + III using the spraying process, as compared to the addition of 25% ethanol as a flow;

and an increase in yield of 0.3 g IgG per liter of plasma (averaged from the range of 0.4 to 0.06 g / l) was achieved, as compared to the addition of 20% ethanol as a flow as It is currently used in manufacturing. 40

IgG yield in the filtrate is significantly higher compared to the reference and well above 80 to 86% currently achieved in manufacturing with the addition of 20% ethanol as a flux in the precipitation of II + III.

C. Fractionation II + III (20% ethanol): the initial pH is maintained throughout the fractionation II + III 5

Example 16

Process

 10

50 liters of plasma were thawed while mixing at 17-20 ° C for 27 hours. Fractionation I was performed as mentioned in the previous sections as the optimized procedure. Supernatant I was separated into two parts:

(1) pH adjustment in the worst case: pH adjustment before and after the addition of ethanol but not during the incubation period.

(2) Optimized pH adjustment: pH adjustment before and after the addition of ethanol and also pH readjustment during retention time. The solution was constantly stirred during the retention time.

 twenty

The pH of the supernatant of I in both parts was adjusted to 6.7 before the addition of ethanol using a buffer of pH 4. Ethanol was added by spraying and the pH was readjusted to 6.9 after the addition of ethanol.

In part (1) the pH adjustment was carried out with less care to simulate the worst case. The pH of the solution was adjusted directly after the addition of ethanol but not during the incubation. In part (2) the pH was readjusted to a constant value of 6.8 to 7.0 during the incubation time of 10 hours.

Results

The IgG was measured nephelometrically again and is shown in Table 16. By constant pH readjustment at a constant value of approximately 6.9 during the retention time, only 0.13 g IgG per liter of 30 was lost. plasma compared to an average of 0.4 g / l plasma in large-scale manufacturing. A yield increase of 0.07 g IgG per liter of plasma was achieved compared to the reference (no spraying but with constant stirring during the retention period). A yield increase of approximately 0.20 g to approximately 0.30 g IgG per liter of plasma was achieved compared to the conventional procedure currently in use (loss of 0.38 g IgG per liter of plasma, see Table 13) . 35

Table 16

 IgG volume measured by nephelometrically

 Sample kg Yield (%) g / l plasma

 Reservation

 Plasma 45.20 100.00 4.90

 Fraction I

 Supernatant I 68.66 102.61 5.03

 Optimized pH adjustment

 Supernatant II + III 53.09 0.26 0.13

 Reference

 Supernatant II + III 52.69 0.41 0.20

conclusion

 40

Loss of IgG in the supernatant of II + III is reduced from the current level of 0.4 g IgG / l plasma in manufacturing batches at a level of 0.13 g / l plasma in precipitation with 20% ethanol, and at a level of less than 0.08 g / l plasma in precipitation with 25% ethanol when ethanol is added by spraying and a continuous pH of 6.9 ± 0.1 is maintained during precipitation.

 Four. Five

In precipitation I, the addition of ethanol before the adjustment of pH by spraying results in an increase in the IgG yield of 0.1 to 0.2 g / l plasma in the fractionation supernatant I.

Analysis

 fifty

IgG was measured nephelometrically in all experiments and may have a variance of at least - / + 5.0% (as indicated by the nephelometer manufacturer, Siemens AG). Therefore, it is possible that the increase in actual yield obtained by the improved process during manufacturing may be slightly smaller or greater than that indicated in the examples.

 55

As additional evidence of the increase in yield by the new and improved procedure, the IgG weight of the precipitate was compared with the average IgG weight of the precipitate obtained from the same plasma source in the manufacturing. 18 kg of precipitated IgG per 1000 liters are obtained from the Cohn's reserve from the source by the procedure currently used in manufacturing, unlike the pilot scale study (section B above) in which 20.8 kg were obtained of precipitated IgG (20% ethanol and optimized pH adjustment in fractionation II + III, 5 buffer addition and spray ethanol). This is an increase of more than 2 kg of precipitate IgG per 1000 liters of Cohn reserve.

Example 17

 10

This example demonstrates that the addition of a combustion silica treatment step prior to filtration of the suspension of fraction II + III results in higher purity IgG filtrates. In summary, plasma devoid of cryoprecipitate was fractionated as described above to the phase of fraction II + III, at which point it was divided into two samples. The first sample was clarified only by the addition of filter aid to the filtration of the standard fraction II + III suspension (Figure 7A). The second sample was subjected to the pretreatment of fumed silica, as described herein, prior to the addition of the filter aid and filtration of the standard fraction II + III suspension (Figure 7B).

Next, the protein components were separated from those filtered by cellulose acetate electrophoresis and the areas of the individual peaks were calculated using standard procedures. As can be seen in the 20 chromatographs and quantified data, the second sample, which was treated with combustion silica before filtration, resulted in filtering with a purity of IgG much greater than the sample not treated with combustion silica ( 68.8% versus 55.7 of γ-globulin; compare table 18 with table 16).

Table 17. Quantification of protein peaks separated by cellulose acetate electrophoresis from the suspension of fraction II + III clarified by the addition of filter aid only before filtration, shown in Figure 7A.

 Peak number from left to right

 area (%)

 fraction

 one

 55.7 y-globulin

 2

 3.2 denatured protein

 3

 3.7 y-globulin

 4

 25.5 α / β-globulin

 5

 11.9 albumin

Table 18. Quantification of protein peaks separated by cellulose acetate electrophoresis of the suspension of fraction II + III pretreated with combustion silica and rinsed by the addition of filter aid before filtration, shown in Figure 7B.

 Peak number from left to right

 area (%)

 fraction

 one

 68.8 y-globulin

 2

 18.9 α / β-globulin

 3

 12.3 albumin

Example 18

 35

The present example illustrates the ultrafiltration and formulation of a 20% IgG preparation suitable for subcutaneous administration. This information was collected during the production of 20% scaled and preclinical IgG preparations. The procedure used for the manufacture of 20% batches before the nanofiltration stage was as described above. Ultra- / diafiltration was improved to concentrate the solution to 20%. To reduce the loss in performance to a minimum, the post-wash of the ultrafiltration device used for diafiltration is concentrated by a second smaller device equipped with the same membranes and then added to the bulk solution.

Surprisingly, it was shown that viral inactivation during storage at low pH is not influenced by the protein concentration of the solution. A similar viral reduction was achieved both in the solution at 45

10% (GAMMAGARD® LIQUID) as in the 20% solution. Therefore, storage was maintained at low pH as a viral reduction step for the product at 20%.

Prior to nanofiltration, the glycine concentration of the IgG solution is adjusted to a target of 0.25 M. The solution is then concentrated to a protein concentration of 6 ± 2% w / w through ultrafiltration. (PHEW). The pH is adjusted to 5.2 ± 0.2. The UF membrane used has a nominal molecular weight limit (NMWCO) of 50,000 daltons or less and is especially designed for high viscosity products (for example, Millipore V Screen).

Then, the concentrate is diafiltered against 0.25 M glycine solution, pH 4.2 ± 0.2. The minimum exchange volume is 10 times the volume of the original concentrate. Throughout the ultrafiltration / diafiltration operation, the solution is maintained at approximately 4 ° C to 20 ° C.

After diafiltration, the solution is concentrated to a protein concentration of at least 22% (w / v). The temperature of the solution is adjusted from 2 ° C to 8 ° C.

 fifteen

To recover the complete residual protein in the system, the post-wash of the first largest ultrafiltration system is carried out with at least 2 times the dead volume in the recirculation mode to ensure that all the protein is washed away. Next, the post-wash of the first ultrafiltration system is concentrated to a protein concentration of at least 22% w / v with a second ultra- / diafiltration system equipped with the same type of membrane that is sized one tenth or less than the first. Postwash concentrate 20 is added to the bulk solution. The second ultrafiltration system is then post-washed. This postwash is used for adjusting the protein concentration of the final formulation. The temperature of the solution is maintained between approximately 2 ° C and 8 ° C.

To formulate the final solution, the protein concentration is adjusted to approximately 20.4 ± 0.4% (w / v) with post-wash of the second smallest ultrafiltration system and / or with diafiltration buffer. The pH is adjusted to between approximately 4.4 to 4.9, if necessary.

Example 19

 30

To compare the fraction of IgG recovered in the filtrate of fraction II + III in the current GAMMAGARD® LIQUID manufacturing process, five purifications of IgG were made at manufacturing scale using improved precipitation and dissolution procedures of fraction II + III provided in this document. In summary, the precipitation of Cohn's IgG reserves with a starting IgG concentration of approximately 6.14 g / l at -7 ° C with 25% ethanol incorporated by the addition of flux, compared with -5 ° C and 35 20% ethanol, as used in the current manufacturing process. Then, the precipitate of fraction II + III modified with a solution buffer with a pH of 4.3 or adjusted with 0.06% glacial acetic acid was extracted and subsequently filtered through a filter deep with a final wash of 4.3 volumes of dead filter buffer solution. As seen in Table 18, the filtered fraction II + III modified prepared according to the improved procedures provided herein contained a significantly higher percentage (an increase of at least 8.0%) of the IgG. present in the initial Cohn reserve than that of the filtrate of fraction II + III prepared in accordance with this manufacturing procedure (91.1% and 91.6% versus 83.1% and 83.8%, respectively).

Table 18. Average recovery of IgG in the filtration of fraction II + III of all Baxter 45 manufacturing cycles carried out in Vienna during 2008 and part of 2009, compared to the recovery of IgG in batches manufactured in accordance with the Enhanced procedures provided in this document.

 Process

 Manufacture in Vienna R and D Scaling **

 Current

 2008

 IgG in Cohn reserve (g / l)

 6.26 6.31 6.14 6.13

 Alcohol in precip. II + III (%)

 20 20 25 25

 ml acetic acid / 1000 l of dissolution buffer

 510 510 Solution at pH 4.3; change to 4.9 before filtration 600

 Aerosil (g / g paste)

 0.045 0.04 0.04 0.04

 Post-wash in dead filter-press volumes

 1.8 1.8 4.3 4.3

 Average IgG in filtering (% Cohn reserve)

 83.1% 57 lots 83.8% 200 lots 91.1% 3 lots 91.6% 2 lots

 Additional IgG in the filtrate

 n.a. n.a. + 8.0% + 8.5%

Example 20

To determine the purity of the IgG compositions provided herein, three batches of IgG were prepared in accordance with the improved procedures provided herein. The final IgG products of these purifications were then tested for various contaminants, including 5 IgA, IgM, amidolytic activity, C3, and fibrinogen, as well as to determine the percentage of IgG monomers / dimers in the final composition. As can be seen in Table 19 below, the improved manufacturing procedures provided herein result in final bulk compositions with an increase in IgG recovery, from 73.6% to 78.5% of the Starting material compared to 60% to 70% for procedures used today, while maintaining purity profiles 10 that are as good, or better, than current IgG manufacturing standards.

Table 19. Impurity levels and composition of IgG monomers / dimers in the IgG composition prepared in accordance with the improved manufacturing procedures provided herein.

 fifteen

 Option

 1 3 6

 Experiment No. (P0..10NG2)

 19 20 21

 ANX continuous flow protein yield [g / l plasma]

 4.35 4.35 4.38

 IgG recovery: Cohn's starting material with respect to the bulk [%]

 73.6 78.5 78.5

 Final Bulk Characterization

 IgA [μg / ml with 10% protein]

 29 26 pend.

 IgM [μg / ml with 10% protein]

 1.1 1.1 pend.

 amidolytic activity [PL-1 nmol / ml min]

 pend. <10 <10

 MSD monomers / dimers [%]

 99.7 99.9 99.7

 C3 [μg / ml with 10% protein]

 pend. pend. pend.

 Fibrinogen [μg / ml with 10% protein]

 <0.2 pend. pend.

Claims (36)

1. A process for preparing an enriched IgG composition from plasma, the process comprising the steps of:  5 (a) precipitate a plasma fraction devoid of cryoprecipitate, in a first stage of precipitation, with alcohol between about 6% and about 10% at a pH between about 7.0 and about 7.5 to obtain a first precipitate and a first supernatant; (b) precipitate the IgG of the first supernatant, in a second stage of precipitation, with alcohol between about 20% and about 25% at a pH between about 6.7 and about 7.3 to form a second precipitate; (c) resuspend the second precipitate to form a suspension;  fifteen (d) precipitate the IgG of the suspension formed in step (c), in a third stage of precipitation, with alcohol between about 22% and about 28% at a pH between about 6.7 and about 7.3 to form a third precipitate; (e) resuspend the third precipitate to form a suspension; and 20 (f) separating the soluble fraction from the suspension formed in step (e), thereby forming an enriched IgG composition, wherein at least one of the first stage of precipitation, second of precipitation, or third stage of precipitation 25 comprises the spray addition of the alcohol. 2. The method of claim 1, wherein all of the first precipitation stage, second precipitation stage, and third precipitation stage comprise the alcohol addition by spraying.  30 3. The method of claim 1 or 2, wherein the pH of at least one of the first precipitation stage, second precipitation stage, or third precipitation stage is achieved by the addition of a pH modifying solution after The addition of alcohol. 4. The method of any one of claims 1 to 3, wherein the pH of all of the first stage of precipitation, second stage of precipitation, and third stage of precipitation is achieved by the addition of a pH modifying solution after the addition of alcohol. 5. The method of claim 3 or 4, wherein the addition of a pH modifying solution comprises the spray addition of the pH modifying solution. 40 6. The method of any one of claims 3 to 5, wherein the pH of the precipitation step is modified before and after the alcohol addition, during and after the alcohol addition, or before, during and after of the addition of alcohol.  Four. Five 7. The method of any one of claims 1 to 6, wherein the pH of at least one precipitation stage is maintained throughout the precipitation stage by continuous pH adjustment. 8. The method of any one of claims 1 to 7, wherein the method further comprises an ion exchange chromatography purification step. fifty 9. The method of claim 8, wherein the method comprises both an anion exchange chromatography purification step and a cation exchange chromatography purification step. 10. The method of any one of claims 1 to 9, wherein the method further comprises a nanofiltration stage and / or an ultrafiltration / diafiltration stage. 11. The method of any one of claims 1 to 10, wherein the enriched IgG composition obtained in step (f) contains at least 85%, preferably at least 90% of the IgG content found in the fraction of plasma devoid of cryoprecipitate used in step (a). 60 12. The method of any one of claims 1 to 11, wherein the purity of the γ-globulins in the final IgG composition is at least about 95%, preferably at least about 98%, more preferably at minus about 99%.  65 13. The method of claim 1, wherein the method comprises the steps of: (a) adjust the pH of a plasma fraction devoid of cryoprecipitate to approximately 7.0; (b) adjust the ethanol concentration of the plasma fraction devoid of cryoprecipitate from step (a) to about 25% (v / v) at a temperature between about -7 ° C and about -9 ° C, forming it mode a mixture; 5 (c) separating liquid and precipitate from the mixture of step (b); (d) resuspend the precipitate from step (c) with a buffer containing phosphate and acetate, in which the pH of the buffer is adjusted with between 300 ml and 700 ml of glacial acetic acid per 1000 l of buffer, forming this mode a suspension; (e) mix finely divided silicon dioxide (SiO2) with the suspension of step (d) for at least about 30 minutes;  fifteen (f) filter the suspension with a filter press, thus forming a filtrate; (g) wash the filter press with at least 3 dead volumes of the filter press of a buffer containing phosphate and acetate, in which the pH of the buffer is adjusted with between 50 ml and 200 ml of glacial acetic acid per 1000 l buffer, thus forming a wash solution; twenty (h) combining the filtrate of step (f) with the washing solution of step (g), thereby forming a solution, and treating the solution with a detergent; (i) adjust the pH of the solution of step (h) to about 7.0 and add ethanol to a final concentration of about 25%, thereby forming a precipitate; (j) separating liquid and precipitate from the mixture of step (i); (k) dissolve the precipitate in an aqueous solution comprising a solvent or detergent and hold the solution for at least 60 minutes; (l) passing the solution after step (k) through a cation exchange chromatography column and eluting the absorbed proteins in the column in an eluate;  35 (m) passing the eluate from step (1) through an anion exchange chromatography column to generate an effluent; (n) passing the effluent from step (m) through a nanofilter to generate a nanofiltrate;  40 (o) passing the nanofiltrate of step (n) through an ultrafiltration membrane to generate an ultrafiltrate; Y (p) diafiltration the ultrafiltrate of step (o) against a diafiltration buffer to generate a diafiltrate having a protein concentration of between about 8% (w / v) and about 12% (w / v ), thereby obtaining a concentrated IgG composition. Four. Five 14. The method of claim 13, wherein the concentration of ethanol is adjusted in at least one of steps (b) and (i) by introducing ethanol into the spray fraction. 15. The method of claim 13 or 14, wherein the pH is adjusted to the appropriate pH after the addition of ethanol in at least one of steps (b) and (i). 16. The method of claim 15, wherein the pH is maintained for the entire precipitation reaction by the continuous adjustment of the pH in at least one of the steps (b) and (i).  55 17. The method of any one of claims 13 to 16, wherein the pH is adjusted in at least one stage by spraying a solution that can adjust the pH. 18. The method of claim 17, wherein the pH is adjusted by spraying glacial acetic acid.  60 19. The method of any one of claims 13 to 18, wherein the method further comprises filtering the diafiltrate through a filter of 0.22 µm or less. 20. The method of any one of claims 13 to 19, wherein the plasma is human plasma.  65 21. The method of any one of claims 13 to 20, wherein the plasma fraction devoid of cryoprecipitate is formed by a method comprising the steps of: (i) cooling a mixture of combined plasma donations to a temperature between about 2 ° C and about 10 ° C; 5 (ii) separating liquid and precipitate from the mixture of step (i) by centrifugation; (iii) adding ethanol in the liquid supernatant formed in step (ii) to a final concentration of ethanol between about 5% (v / v) and about 10% (v / v), thereby forming a mixture; 10 (iv) cooling the mixture formed in step (iii) to between about -4 ° C and about 0 ° C; (v) separating liquid and precipitate from the mixture of step (iv) by centrifugation; and isolating the supernatant, thereby forming a plasma fraction devoid of cryoprecipitate. fifteen 22. The process of any one of claims 13 to 21, wherein the temperature of the mixture of step (b) is approximately -7 ° C. 23. The method of any one of claims 13 to 22, wherein the precipitate formed in step (c) 20 is resuspended with between about 12 l and about 18 l of buffer per kg of precipitate, preferably with about 15 l of buffer per kg of precipitate. 24. The method of any one of claims 13 to 23, wherein the amount of silicon dioxide added to the suspension in step (e) is between about 0.02 grams per gram of precipitate formed in step ( c) and about 0.06 grams per gram of precipitate formed in step (c), preferably in which the amount of silicon dioxide added to the suspension in step (e) is about 0.04 grams per gram of precipitate formed in step (c). 25. The method of any one of claims 13 to 24, wherein a filter aid is added to the mixture before filtration in step (f). 26. The method of claim 25, wherein the filter aid is diatomaceous earth. 27. The method of any one of claims 13 to 26, wherein the purity of the γ-globulins in the filtrate formed in step (f) is at least about 85%. 28. The method of any one of claims 13 to 27, wherein the filtrate formed in step (f) contains less than about 10 mg of fibrinogen per gram of total protein.  40 29. The method of any one of claims 13 to 28, wherein the filtrate formed in step (f) contains less than about 500 IU of PKA activity per gram of total protein. 30. The method of any one of claims 13 to 29, wherein the detergent used in step (h) comprises polysorbate-80 between about 0.1% (w / v) and about 0.3% ( p / v). Four. Five 31. The method of any one of claims 13 to 30, wherein the aqueous solution used in step (k) comprises Triton-X 100, polysorbate-80 and TNBP. 32. The method of any one of claims 13 to 31, wherein the solution is maintained in step 50 (k) at a temperature between about 18 ° C and about 25 ° C. 33. The method of any one of claims 13 to 32, wherein the nanofilter of step (n) has an average pore size between about 15 nm and about 72 nm, preferably between about 19 nm and about 35 nm, more preferably about 35 nm. 55 34. The method of any one of claims 13 to 33, wherein the ultrafiltration membrane of step (o) has a nominal molecular weight limit (NMWCO) of approximately 100 kDa or less. 35. The method of any one of claims 13 to 34, wherein the protein concentration of the composition is approximately 10% (w / v). 36. The method of any one of claims 13 to 35, wherein the solution formed in step (h) contains at least about 85%, preferably at least about 90% of the IgG present in the plasma fraction devoid of cryoprecipitate from stage (a). 65